Compound that specifically binds to kir3dl2 for use in the treatment of peripheral t cell lymphoma

ABSTRACT

The present invention relates to methods for the treatment, prevention and diagnosis of peripheral T cell lymphoma using compounds that specifically bind KIR3DL2. The invention also relates to use of antibodies that specifically bind KIR3DL2 in diagnostic and theranostic assays in the detection and treatment of peripheral T cell lymphoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/623,572, filed Jun. 15, 2017, now U.S. Pat. No. 10,174,112, which isa divisional of U.S. application Ser. No. 14/769,158, filed Aug. 20,2015, now abandoned, which is the U.S. national stage application ofInternational Patent Application No. PCT/EP2014/053340, filed Feb. 20,2014, which claims the benefit of U.S. Provisional Application No.61/766,798, filed Feb. 20, 2013, and U.S. Provisional Application No.61/831,809, filed Jun. 6, 2013, the disclosures of which areincorporated herein by reference in their entirety, including anydrawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“KIR-4 PCT_ST25”, created 20 Feb. 2014, which is 39 KB in size. Theinformation in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the use of KIR3DL2-targeting agents for thediagnosis and treatment of aggressive lymphomas.

BACKGROUND OF THE INVENTION

Peripheral T cell non-Hodgkin's lymphomas (PTCLs) account for 15% to 20%of aggressive lymphomas and for 7% to 10% of all the non-Hodgkin'slymphomas (NHLs) in Western countries. They usually occur in middle-agedto elderly patients, and presenting features are characterized by adisseminated disease in 68% of the patients, with systemic symptoms innearly half of them (45%), bone marrow (BM) involvement in a quarter(25.8%), and extranodal disease in a third (37%). Despite aggressivetherapy, more than half the patients die of their disease. While certaindistinctive disease entities have improved prognostics if treated, theprognosis for many aggressive PTCLs remains relatively unchanged by theuse of second- and third-generation chemotherapy regimens and 5-yearoverall survival (OS) still remains between 25% and 47% for PTCL-NOS,for example.

Consequently, there is a need in the art for improved benefit topatients having PTCL.

SUMMARY OF THE INVENTION

The present inventors have discovered that KIR3DL2 is expressed on thesurface of peripheral T cell lymphomas (PTCLs), particularly advancedand/or aggressive PTCLs. In KIR3DL2-positive PTCLs, membranar KIR3DL2expression permits targeting with KIR3DL2-binding antibodies (e.g., asassessed by immunohistochemistry). KIR3DL2 is expressed on few othertissues (only on a small fraction of healthy NK and T cells), permittingKIR3DL2 to serve as a marker and target for the detection and treatmentof peripheral T cell lymphomas, particularly aggressive and/or advancedT cell lymphomas, e.g., aggressive and/or advanced nodal or extranodalperipheral T cell lymphomas. Accordingly, in one embodiment, a method isprovided for treating or preventing a peripheral T cell lymphoma in anindividual, the method comprising administering to an individual atherapeutically active amount of a compound that binds a KIR3DL2polypeptide. In one aspect a compound is provided that binds a KIR3DL2polypeptide, for use in the treatment or prevention of peripheral T celllymphomas. In one embodiment, a method is provided for treating anindividual having an advanced (e.g., stage IV or more) peripheral T celllymphoma, the method comprising administering to an individual atherapeutically active amount of a compound that binds a KIR3DL2polypeptide. In one aspect the compound that binds a KIR3DL2 polypeptideis capable of depleting a cell that expresses KIR3DL2 at its surface,e.g., a PTCL cell that expresses KIR3DL2 on its surface. In one aspectthe compound is a depleting anti-KIR3DL2 antibody. In one embodiment acompound is provided that binds a KIR3DL2 polypeptide and depletesKIR3DL2-expressing tumor cells, for use in the treatment or preventionof a PTCL in an individual. Optionally the treatment or preventioncomprises administration of the compound that binds a KIR3DL2polypeptide to an individual having a PTCL. In one embodiment of any ofthe therapeutic uses or PTCL treatment or prevention methods herein, theindividual has an ortho-visceral extranodal PTCL, optionally wherein theortho-visceral extranodal PTCL is a NK/T− lymphoma or anenteropathy-associated T cell lymphoma (EATL). In one embodiment of anyof the therapeutic uses or PTCL treatment or prevention methods herein,the individual has an anaplastic large cell lymphoma (ALCL). In oneembodiment of any of the therapeutic uses or PTCL treatment orprevention methods herein, the individual has a PTCL-NOS. In oneembodiment, the treatment or prevention of a PTCL in an individualcomprises:

a) determining the KIR3DL2 polypeptide status of malignant cells withinthe individual having a PTCL, and

b) upon a determination that the individual has KIR3DL2 polypeptidesprominently expressed on the surface of malignant cells, administeringto the individual said compound that binds a KIR3DL2 polypeptide.

Additionally, antibodies are provided that are particularly effective indiagnostic or prognostic assays to detect KIR3DL2 expression on tumorcells, notably in immunohistochemistry assays. The antibodies arecapable of detecting membranar KIR3DL2 in cases where prior antibodieswere not able to detect such KIR3DL2 expression, including withspecificity over KIR3DL1.

In another embodiment a method is provided comprising a KIR3DL2detection step to identify patients having KIR3DL2+ tumors; thesepatients can thereafter be treated with a KIR3DL2-binding agent. Suchmethod permits KIR3DL2 therapy to be more precisely directed to patientswithout reliance on disease staging. Such method also helps permit theprevention of advanced PTCL (e.g., prevention of progressing of PTCL toan advanced stage, e.g., stage IV) because patients can be treated asKIR3DL2 appears.

In a further aspect, it has been found that patients withKIR3DL2-positive PTCL-NOS can have tumors that are CD30-negative (tumorcells do not express CD30 on their surface). Thus, methods are providedof treating a CD30-negative PTCL, e.g., a PTCL-NOS, comprisingadministering a compound that binds a KIR3DL2 polypeptide to a patienthaving CD30-negative PTCL. In another embodiment of treating anindividual having a PTCL, the methods or uses comprise administering acompound that binds a KIR3DL2 polypeptide to an individual having a PTCLwho is refractive to treatment with an anti-CD30 antibody. In otherembodiments, when PTCLs are CD30-positive (e.g., anaplastic large celllymphomas which broadly express CD30, certain PTCL-NOS), a compound thatbinds a KIR3DL2 polypeptide can be administered in combination with ananti-CD30 antibody (e.g., a depleting anti-CD30 antibody).

In one embodiment, provided is a method for detecting a peripheral Tcell lymphoma in an individual, the method comprising detecting aKIR3DL2 nucleic acid or polypeptide in a biological sample (e.g., on acell) from an individual. In one embodiment, a method is provided fordetecting an aggressive or advanced (e.g., stage IV or higher)peripheral T cell lymphoma in an individual, the method comprisingdetecting a KIR3DL2 nucleic acid or polypeptide in a biological sample(e.g., on a cell) from an individual. A determination that a biologicalsample expresses KIR3DL2 indicates that the patient has a peripheral Tcell lymphoma (or advanced/aggressive PTCL). In one embodiment, themethod comprises determining the level of expression of a KIR3DL2nucleic acid or polypeptide in a biological sample and comparing thelevel to a reference level (e.g., a value, weak cell surface staining,etc.) corresponding to a healthy individual. A determination that abiological sample expresses a KIR3DL2 nucleic acid or polypeptide at alevel that is increased compared to the reference level indicates thatthe patient has a peripheral T cell lymphoma. Optionally, detecting aKIR3DL2 polypeptide in a biological sample comprises detecting a KIR3DL2polypeptide expressed on the surface of a malignant lymphocyte.

In one embodiment, a method is provided comprising:

(a) determining whether an individual has an advanced and/or aggressiveperipheral T cell lymphoma (e.g., stage IV); and(b) if the individual has an advanced and/or aggressive peripheral Tcell lymphoma, treating the individual with a therapeutically activeamount of a compound that binds a KIR3DL2 polypeptide.

In one embodiment, a method is provided comprising: (a) determiningwhether an individual has a peripheral T cell lymphoma; and (b) if theindividual has a peripheral T cell lymphoma, determining whether theindividual has peripheral T cell lymphoma cells that express a KIR3DL2polypeptide. The method may optionally further comprise treating theindividual with a therapeutically active amount of a compound that bindsa KIR3DL2 polypeptide if the individual has peripheral T cell lymphomacells that express KIR3DL2 on their surface.

In one embodiment, a method is provided comprising:

(a) determining whether an individual has peripheral T cell lymphomacells that express a KIR3DL2 polypeptide on their surface; and(b) if the individual has peripheral T cell lymphoma cells that expressKIR3DL2 on their surface, treating the individual with a therapeuticallyactive amount of a compound that binds a KIR3DL2 polypeptide.

In one embodiment, a method is provided comprising treating anindividual having a CD30-negative PTCL. In one embodiment, the methodcomprises:

(a) determining whether an individual (e.g., an individual with advancedPTCL) has peripheral T cell lymphoma cells that express CD30 on theirsurface, and optionally further determining whether the individual hasperipheral T cell lymphoma cells that express KIR3DL2 on their surface;and(b) if the individual has peripheral T cell lymphoma cells that do notexpress CD30 on their surface, optionally wherein the individual hasperipheral T cell lymphoma cells that express a KIR3DL2 polypeptide ontheir surface, treating the individual with a therapeutically activeamount of a compound that binds a KIR3DL2 polypeptide.

In one embodiment, a method is provided comprising treating anindividual having a CD30-positive PTCL. In one embodiment, the methodcomprises:

(a) determining whether an individual (e.g., an individual with advancedPTCL) has peripheral T cell lymphoma cells that express CD30 on theirsurface, and further determining whether the individual has peripheral Tcell lymphoma cells that express KIR3DL2 on their surface; and(b) if the individual has peripheral T cell lymphoma cells that expressCD30 on their surface, and the individual has peripheral T cell lymphomacells that express a KIR3DL2 polypeptide on their surface, treating theindividual with a therapeutically active amount of a compound that bindsa KIR3DL2 polypeptide and with a therapeutically active amount of ananti-CD30 antibody.

In one embodiment of any of the methods, determining whether anindividual has peripheral T cell lymphoma cells that express a KIR3DL2polypeptide comprises obtaining a biological sample from the individualthat comprises peripheral T cell lymphoma cells, bringing said cellsinto contact with an antibody that binds a KIR3DL2 polypeptide, anddetecting whether the cells express KIR3DL2 on their surface.

Optionally, in any embodiment, determining whether an individual hasperipheral T cell lymphoma cells that express KIR3DL2 comprisesconducting an immunohistochemistry assay, e.g., an immunohistochemistryassay comprising obtaining from an individual a biological sample thatcomprises tumor cells, fixing and sectioning said sample to obtain atissue section, bringing said tissue section into contact with anantibody (e.g., an antibody that competes with antibody 12B11 forbinding to a human KIR3DL2 polypeptide) and detecting expression ofKIR3DL2 (e.g., detecting cells that express KIR3DL2). In one embodiment,the tissue section is a frozen tissue section. Optionally determiningwhether an individual has peripheral T cell lymphoma cells that expressKIR3DL2 comprises conducting a flow cytometry assay. Both IHC and flowcytometry can detect surface expression of KIR3DL2.

Also provided is a method of treating a patient with a PTCL, the methodcomprising: a) determining the KIR3DL2 polypeptide status of malignantcells (e.g., PTCL cells) within the patient, e.g., determining whether aKIR3DL2 polypeptide is prominently expressed on the surface of saidmalignant cells, and b) administering a compound to the patient thatspecifically binds to a KIR3DL2 polypeptide that is prominentlyexpressed in said malignant cells (e.g., prominently expressed on thesurface of malignant cells). Optionally, determining the KIR3DL2polypeptide status comprises determining whether a KIR3DL2 polypeptideis prominently expressed on the surface of said malignant cells.Optionally, determining whether a KIR3DL2 polypeptide is prominentlyexpressed on the surface of said malignant cells comprises obtainingfrom the individual a biological sample that comprises peripheral T celllymphoma cells, bringing said cells into contact with an antibody thatbinds a KIR3DL2 polypeptide, and detecting cells that express KIR3DL2(e.g., determining the number or portion of cells that express KIR3DL2).

Preferably the compound that binds a KIR3DL2 polypeptide is a compoundthat causes the death of a KIR3DL2-expressing cell. Optionally, thecompound that binds a KIR3DL2 polypeptide is a polypeptide, optionallyan antibody (e.g., a monoclonal antibody), that binds a KIR3DL2polypeptide, optionally a polypeptide or other compound that is anatural ligand of NKp46. Optionally, the antibody is a depletingantibody. Optionally, the antibody is an antibody that directs ADCCand/or CDC toward a KIR3DL2-expressing cell. Optionally, the antibody isan antibody that delivers a cytotoxic agent (e.g., a small molecule) toa KIR3DL2-expressing cell.

In one embodiment, the antibody used in any embodiment herein binds aKIR3DL2 polypeptide, optionally wherein the antibody does notsubstantially bind to a KIR3DL1 polypeptide and has bivalent bindingaffinity (K_(D)) for a human KIR3DL2 polypeptide of less than 10⁻⁸ M. Inone embodiment, the antibody binds a KIR3DL2 polypeptide in its D1domain. In one embodiment, the antibody binds a KIR3DL2 polypeptide,wherein said antibody does not substantially bind to a KIR3DL1polypeptide, and wherein said antibody binds to at least one residue inthe segment corresponding to residues 99-192 of the mature KIR3DL2polypeptide of SEQ ID NO: 1.

In one embodiment, the antibody used herein competes for binding to aKIR3DL2 polypeptide with an antibody selected from the group consistingof:

(a) an antibody having respectively a VH and VL region of SEQ ID NOS: 5and 6 (19H12);

(b) an antibody having respectively a VH and VL region of SEQ ID NOS: 16and 17 (12B11); or

(c) an antibody having respectively a VH and VL region of SEQ ID NOS: 33and 34 (2B12).

Optionally, the antibody binds an epitope comprising residue P179 and/orresidue S181 of the KIR3DL2 polypeptide of SEQ ID NO: 1, and/or hasreduced binding to a KIR3DL2 polypeptide having a mutation at residueP179 and/or residue S181 of SEQ ID NO: 1, compared to a wild-typeKIR3DL2 polypeptide of SEQ ID NO: 1.

Optionally, the antibody used herein binds an epitope comprisingresidues N99, H100, E130, H131, F132, V178, H180, P182, Y183 and/or Q184of SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2 polypeptidehaving a mutation at residues N99, H100, E130, H131, F132, V178, H180,P182, Y183 and/or Q184 of SEQ ID NO: 1, compared to a wild-type KIR3DL2polypeptide of SEQ ID NO: 1.

Optionally, the antibody (e.g., antibody 2B12 or an antibody thatcompetes therewith for binding to KIR3DL2) binds an epitope comprisingresidue I60 and/or residue G62 of the KIR3DL2 polypeptide of SEQ ID NO:1, and/or has reduced binding to a KIR3DL2 polypeptide having a mutationat residue I60 and/or residue G62 of SEQ ID NO: 1, compared to awild-type KIR3DL2 polypeptide of SEQ ID NO: 1. Optionally, the antibodybinds an epitope comprising residues P14, S15 and/or H23 of the KIR3DL2polypeptide of SEQ ID NO: 1, and/or has reduced binding to a KIR3DL2polypeptide having a mutation at residues P14, S15 and/or H23 of SEQ IDNO: 1, compared to a wild-type KIR3DL2 polypeptide of SEQ ID NO: 1.Optionally, the antibody used herein binds an epitope comprisingresidues 160, G62, P14, S15 and/or H23 of SEQ ID NO: 1.

Optionally, the compound that binds a KIR3DL2 polypeptide isadministered between once daily and once per month. Optionally, thecomposition is administered as monotherapy. Optionally, the compositionis administered in combination with a second therapeutic agent.Optionally, the composition is administered in combination with ananti-cancer agent.

In one embodiment, a method is provided of producing a composition forthe treatment of peripheral T cell lymphoma or for use in the preventionof peripheral T cell lymphoma in a mammalian subject, said methodcomprising the steps of: a) providing a plurality of test compositions;b) testing each compound for the ability to bind KIR3DL2 and/or causethe depletion of KIR3DL2-expressing cells; and c) selecting a compoundwhich binds a KIR3DL2 polypeptide and/or causes the depletion ofKIR3DL2-expressing cells as suitable for the treatment of peripheral Tcell lymphoma or for use in the prevention of peripheral T celllymphoma.

Optionally, the method further comprises producing a quantity of thecompound selected in step c) and/or formulating a quantity of thecompound selected in step c) with a pharmaceutically acceptableexcipient.

Optionally, step b) further comprises testing said test composition forthe ability to direct ADCC and/or CDC toward a KIR3DL2-expressing cell,e.g., a peripheral T cell lymphoma cell.

In one embodiment, provided is a method comprising: (a) determiningwhether an individual has a peripheral T cell lymphoma; and (b) if theindividual has a peripheral T cell lymphoma, treating the individualwith a therapeutically active amount of a compound that binds a KIR3DL2polypeptide.

In one embodiment, the determination of whether an individual has aperipheral T cell lymphoma is made according to standard medicalguidelines.

In one embodiment, determining whether an individual has a peripheral Tcell lymphoma comprises identifying a population of abnormal cells orabnormal numbers of cells. Optionally, said identification is by flowcytometry or immunohistochemistry. Optionally, the method furthercomprises sorting or isolating the population of abnormal cells.

In one embodiment, determining whether an individual has a peripheral Tcell lymphoma comprises detecting cytogenetic aberrations (e.g.,assessing karyotype).

In one embodiment, determining whether an individual has a peripheral Tcell lymphoma comprises sorting the population of abnormal cells, andcontacting nucleic acid isolated from the sorted cells with one or moreoligonucleotides, wherein the contacting determines the presence of aneoplastic genetic marker, thereby detecting the presence of peripheralT cell lymphoma.

In one embodiment, determining whether an individual has a peripheral Tcell lymphoma comprises assessing the levels of a serum protein in theindividual.

Optionally, the method further comprises a step of assessing, followingtreatment with a compound that binds a KIR3DL2 polypeptide, whether theindividual has an amelioration in peripheral T cell lymphoma, e.g.,whether the individual has decreased numbers of peripheral T celllymphoma cells.

In one embodiment of any aspect herein, the PTCL is an aggressive and/oradvanced PTCL. In one embodiment, the PTCL is aggressive non-cutaneousPTCL. In one embodiment, the PTCL is PTCL-NOS (also referred to asPCTL-U). In one embodiment, the PTCL is a nodal (e.g., primarily nodal)PTCL. In one embodiment, the PTCL is an anaplastic large cell lymphoma(ALCL), optionally an ALK-negative ALCL, optionally an ALK-positiveALCL. In one embodiment, the PTCL is an angioimmunoblastic T celllymphoma (AITL), optionally a cutaneous AITL, optionally a non-cutaneousAITL. In one embodiment, a PTCL may be an aggressive, non-cutaneous,primarily nodal PCTL. In one embodiment, the PTCL is an extranodal(e.g., primarily extranodal) PTCL. In one example a PTCL may be anaggressive, non-cutaneous, extranodal PCTL. In one embodiment the PTCLis an ortho-visceral extranodal PTCL. In one embodiment, the PTCL is anextranodal NK/T cell lymphoma, nasal type. In one embodiment, the PTCLis an enteropathy-associated T cell lymphoma. In one embodiment, thePTCL is a hepatosplenic T cell lymphoma, optionally a hepatosplenic αβ Tcell lymphoma, optionally a hepatosplenic γδ T cell lymphoma.

In one embodiment of any aspect herein, the PTCL is a CD30 positive PTCLand the anti-KIR3DL2 antibody is administered in combination with ananti-CD30 antibody. In one embodiment of any aspect herein, the PTCL isa CD4 positive PTCL and the anti-KIR3DL2 antibody is administered incombination with an anti-CD3 antibody.

In one embodiment, a method is provided for diagnosing or monitoring aPTCL in an individual, the method comprising obtaining from anindividual a biological sample that comprises PTCL cells, bringing saidcells into contact with an antibody that binds a human KIR3DL2polypeptide, and detecting cells that express KIR3DL2. Optionally, theantibody that binds a KIR3DL2 polypeptide is an antibody that binds ahuman KIR3DL2 polypeptide but does not bind to a human KIR3DL1polypeptide. Optionally, the antibody that binds a KIR3DL2 polypeptidecompetes with antibody 12B11 for binding to a human KIR3DL2 polypeptide(e.g., antibody 19H12).

In one embodiment, a method is provided for determining whether aKIR3DL2 polypeptide is expressed on the surface of a tumor cell, themethod comprising obtaining from an individual a biological sample thatcomprises tumor cells, bringing said cells into contact with an antibodythat competes with antibody 12B11 for binding to a human KIR3DL2polypeptide (e.g., antibody 19H12), and detecting cells that expressKIR3DL2.

In one embodiment, a method is provided for determining whether aKIR3DL2 polypeptide is expressed on the surface of a cell, the methodcomprising obtaining from an individual (e.g., an individual having aPTCL) a biological sample (e.g., a tissue sample) that comprises tumorcells, fixing and sectioning said sample to obtain a tissue section,bringing said tissue section into contact with an antibody that competeswith antibody 12B11 for binding to a human KIR3DL2 polypeptide (e.g.,antibody 19H12), and detecting expression of KIR3DL2 (e.g., detectingcells that express KIR3DL2). In one embodiment, the tissue section is afrozen tissue section.

In one embodiment, antibodies having advantageous uses in diagnostic andprognostic methods for PTCL and other diseases are provided. A method isprovided comprising:

-   -   (a) obtaining a biological sample of cells and bringing such        cells into contact with antibody 19H12 or a derivative or        fragment thereof, an antibody that competes therewith for        binding to KIR3DL2, or an antibody that binds residue P179        and/or residue S181 on a KIR3DL2 polypeptide, optionally wherein        said antibody is labeled with a detectable moiety; and    -   (b) determining by flow cytometry whether said antibody binds to        said cells, wherein binding indicates that the cells express        KIR3DL2 on their surface.

In another embodiment, a method is provided comprising:

-   -   (a) obtaining a biological sample of cells, preparing frozen        tissue sections from such cells, and bringing such sections into        contact with antibody 12B11 or a derivative or fragment thereof,        an antibody that competes therewith for binding to KIR3DL2, or        an antibody that binds residue P179 and/or residue S181 on a        KIR3DL2 polypeptide, optionally wherein said antibody is labeled        with a detectable moiety; and    -   (b) determining whether said antibody binds to said cells,        wherein binding indicates that the cells express KIR3DL2 on        their surface.

The present disclosure further concerns a method for diagnosing adisease state mediated by pathogenic KIR3DL2-expressing cells, saidmethod comprising the steps of combining with an ex vivo patient samplea composition comprising a conjugate or complex comprises an antibodythat binds specifically to KIR3DL2 expressed on the surface of thepathogenic cells and an imaging agent, and detecting the pathogeniccells that express a receptor for the ligand using flow cytometry.

The present disclosure further concerns a method of determining aprognosis of a cancer by detecting cancer cells in an ex vivo patientsample, said method comprising the steps of: (a) combining with an exvivo patient sample a composition comprising a conjugate or complexcomprises an antibody (e.g., antibody 19H12) that binds specifically toKIR3DL2 expressed on the surface of the pathogenic cells and an imagingagent; (b) detecting the pathogenic cells that express a receptor forthe ligand using flow cytometry; and (c) determining a prognosis for thecancer.

The present disclosure further concerns a method for quantitatingpathogenic cells, said method comprising the steps of: (a) combining,with an ex vivo patient sample, a conjugate or complex which comprises(i) an antibody that binds specifically to KIR3DL2 expressed on thesurface of the pathogenic cells (e.g., antibody 19H12) and (ii) animaging agent; and (b) quantitating said pathogenic cells in the ex vivopatient sample using flow cytometry.

In any of the above flow cytometry-based methods, the antibody binds toa KIR3DL2 polypeptide on the surface of cells but not to a KIR3DL1polypeptide. Optionally, said pathogenic cells are detected by singlephoton flow cytometry. Optionally, said pathogenic cells are detected bymultiphoton flow cytometry. Optionally, the ex vivo patient sample is apatient body fluid. Optionally, the body fluid is selected from thegroup consisting of spinal fluid, lymph fluid, urine, mucus, and blood.Optionally, the pathogenic cells are CD4+ T cells. Optionally, thepathogenic cells are lymphoma cancer cells. Optionally, the cancer cellsare mycosis fungoides and Sézary Syndrome cancer cells. Optionally, theantibody conjugated to an imaging agent is selected from the groupconsisting of anti-KIR3DL2-fluorescein, anti-KIR3DL2-Oregon Green,anti-KIR3DL2-rhodamine, anti-KIR3DL2-phycoerythrin,anti-KIR3DL2-cys-Texas Red, anti-KIR3DL2-Alexa Fluor, andanti-KIR3DL2-DyLight. Optionally, the imaging agent comprises achromophore. Optionally, the chromophore is a fluorescent chromophore.Optionally, the chromophore comprises a compound selected from the groupconsisting of fluorescein, Oregon Green, rhodamine, phycoerythrin, TexasRed, DyLight 680, and Alexa Fluor 488. Optionally, the methods furthercomprise the step of quantitating the pathogenic cells in the ex vivopatient sample.

In any of the above flow cytometry-based methods, the antibodiesoptionally bind to each of the KIR3DL2 polypeptides having the aminoacid sequence shown in SEQ ID NOS: 1, 27 and 29 (alleles_*002, *001 and*007, respectively). In one embodiment, the antibodies bind to each ofthe KIR3DL2 polypeptides having the amino acid sequence shown in SEQ IDNOS: 27 and 31 (alleles_*001 and *009, respectively). In one embodiment,the antibodies bind to each of the KIR3DL2 polypeptides having the aminoacid sequence shown in SEQ ID NOS: 27, 1, 29 and 31 (alleles_*001, *002,*007 and *009, respectively). In one embodiment, the antibodies bind toeach of the KIR3DL2 polypeptides having the amino acid sequence shown inSEQ ID NOS: 27, 1, 2, 28 and 29 (alleles_*001, *002, *003, *005 and*007, respectively). In one embodiment, the antibodies bind to each ofthe KIR3DL2 polypeptides having the amino acid sequence shown in SEQ IDNOS: 27, 1, 29 and 30 (alleles_*001, *002, *007 and *008, respectively).In one embodiment, the antibodies bind to each of the KIR3DL2polypeptides having the amino acid sequence shown in SEQ ID NOS: 27, 1,2, 28, 29 and 30 (alleles_*001, *002, *003, *005, *007 and *008,respectively).

In any of the above flow cytometry-based methods, the antibody binds anepitope comprising one, two, three, four, five or more of residuesselected from the group consisting of M128, E130, H131, R145, V147,Q149, I150, V178, P179, H180 and S181 (with reference to SEQ ID NO: 1),and/or the antibody may or may not have reduced binding to a KIR3DL2polypeptide having a mutation at a residue selected from the groupconsisting of M128, E130, H131, R145, V147, Q149, I150, V178, P179, H180and S181 (with reference to SEQ ID NO: 1). In any of the above flowcytometry-based methods, the antibody binds an epitope comprisingresidues P179 and/or S181 of the KIR3DL2 polypeptide, and/or has reducedbinding to a KIR3DL2 polypeptide having a mutation at residues P179and/or S181 (with reference to SEQ ID NO: 1, e.g., a P179T, S181Tmutant). In one aspect the antibody binds an epitope comprising residuesV178 and/or H180 of the KIR3DL2 polypeptide, and/or has reduced bindingto a KIR3DL2 polypeptide having a mutation at residues V178 and/or H180(with reference to SEQ ID NO: 1, e.g., a V178A, H1805 mutant). In oneaspect the antibody binds an epitope comprising residues E130, H131and/or R145 of the KIR3DL2 polypeptide, and/or has reduced binding to aKIR3DL2 polypeptide having a mutation at residues E130, H131 and/or R145(with reference to SEQ ID NO: 1, e.g., an E1305, H131S, R145S mutant).In any of the above flow cytometry-based methods, the antibody is anantibody that competes with and/or comprises the heavy and/or lightchain CDRs 1, 2 and/or 3 of antibody 19H12 or 12B11.

These aspects are more fully described in, and additional aspects,features, and advantages will be apparent from, the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows staining of frozen tissue sections from RAJI-KIR3DL2 mousetumor models and RAJI-KIR3DL2 cell lines, using AZ158 antibody (seeWO2010/081890) or antibody 12B11. While AZ158 was negative, tumors werepositive when using antibody 12B11 at the same concentration (5 μg/ml)of antibody (see FIG. 1).

FIG. 2 shows staining of frozen tissue sections from cancer patientspreviously stained with AZ158, re-examined using antibody 12B11.Biopsies that had been KIR3DL2-negative with AZ158 were stained with12B11 (i.e., becoming KIR3DL2-positive).

FIG. 3 shows staining by anti-KIR3DL2 antibody on NK/T lymphoma cells,nasal type. The figure additionally shows that the KIR3DL2-positivecells express CD183 (CXCR3), CD56 and CD54 (ICAM).

DESCRIPTION OF THE INVENTION

The identification expression of KIR3DL2 polypeptides at the surface ofmalignant PTCL cells permits the development of therapeutic agents thatare able to directly and specifically target pathogenic cells, as wellas diagnostic agents that can be used to diagnose PTCL.

Provided are methods of using the antigen-binding compounds; forexample, a method is provided for inhibiting PTCL cell proliferation oractivity, for delivering a molecule to a PTCL cell (e.g., a toxicmolecule, a detectable marker), for targeting, identifying or purifyinga cell, for depleting, killing or eliminating a cell, or for reducingcell proliferation, the method comprising exposing a cell, such as aPTCL cell which expresses a KIR3DL2 polypeptide, to a compound thatbinds a KIR3DL2 polypeptide. It will be appreciated that for thepurposes herein, “cell proliferation” can refer to any aspect of thegrowth or proliferation of cells, e.g., cell growth, cell division, orany aspect of the cell cycle. The cell may be in cell culture (in vitro)or in a mammal (in vivo), e.g., a mammal suffering from PTCL. Alsoprovided is a method for inducing the death of a cell or inhibiting theproliferation or activity of a PTCL cell which expresses a KIR3DL2polypeptide, comprising exposing the cell to an antigen-binding compoundthat binds a KIR3DL2 polypeptide in an amount effective to induce deathand/or inhibit the proliferation of the cell.

Antibodies specific for KIR3DL2 can be used for a range of purposes forthe diagnosis or treatment of PTCL, including purifying KIR3DL2 orKIR3DL2-expressing cells in patients having PTCL, suspected of havingPTCL or susceptible to PTCL, targeting KIR3DL2-expressing cells fordestruction in vivo, or specifically labeling/binding KIR3DL2 in vivo,ex vivo, or in vitro, in cells of patients having PTCL, suspected ofhaving PTCL or susceptible to PTCL, including by methods such asimmunoblotting, IHC analysis, i.e., on frozen biopsies, FACS analysis,and immunoprecipitation.

As used herein, “a” or “an” may mean one or more. As used in theclaim(s), when used in conjunction with the word “comprising”, the words“a” or “an” may mean one or more than one. As used herein, “another” maymean at least a second or more.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of” or by “consisting of”.

Whenever within this whole specification “treatment of PTCL” or the likeis mentioned with reference to an anti-KIR3DL2 binding agent (e.g., anantibody), there is meant: (a) the method of treatment of PTCL, saidmethod comprising the step of administering (for at least one treatment)an anti-KIR3DL2 binding agent, (e.g., in a pharmaceutically acceptablecarrier material) to a warm-blooded animal, especially a human, in needof such treatment, in a dose that allows for the treatment of PTCL (atherapeutically effective amount), e.g., in a dose (amount) as specifiedhereinabove and hereinbelow; (b) the use of an anti-KIR3DL2 bindingagent for the treatment of PTCL, or an anti-KIR3DL2 binding agent foruse in said treatment (especially in a human); (c) the use of ananti-KIR3DL2 binding agent for the manufacture of a pharmaceuticalpreparation for the treatment of PTCL, or a method of using ananti-KIR3DL2 binding agent for the manufacture of a pharmaceuticalpreparation for the treatment of PTCL, comprising admixing ananti-KIR3DL2 binding agent with a pharmaceutically acceptable carrier,or a pharmaceutical preparation comprising an effective dose of ananti-KIR3DL2 binding agent that is appropriate for the treatment ofPTCL; or (d) any combination of a), b), and c), in accordance with thesubject matter allowable for patenting in a country where thisapplication is filed.

The term “biopsy” as used herein is defined as removal of a tissue forthe purpose of examination, such as to establish diagnosis. Examples oftypes of biopsies include by application of suction, such as through aneedle attached to a syringe; by instrumental removal of a fragment oftissue; by removal with appropriate instruments through an endoscope; bysurgical excision, such as of the whole lesion; and the like.

The term “antibody” as used herein refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms “variable light chain (V_(L))” and“variable heavy chain (V_(H))” refer to these light and heavy chains,respectively. The heavy-chain constant domains that correspond to thedifferent classes of immunoglobulins are termed “alpha”, “delta”,“epsilon”, “gamma” and “mu”, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. IgG is the exemplary class of antibodies employed hereinbecause they are the most common antibodies in the physiologicalsituation and because they are the most easily made in a laboratorysetting. In one embodiment, an antibody is a monoclonal antibody.Provided are humanized, chimeric, human, or otherwise human-suitableantibodies. “Antibodies” also includes any fragment or derivative of anyof the herein described antibodies.

The term “specifically binds to” means that an antibody can bind in acompetitive binding assay to the binding partner, e.g., KIR3DL2, asassessed using either recombinant forms of the proteins, epitopesthereof, or native proteins present on the surface of isolated targetcells. Competitive binding assays and other methods for determiningspecific binding are further described below and are well-known in theart.

When an antibody is said to “compete with” a particular monoclonalantibody, it means that the antibody competes with the monoclonalantibody in a binding assay using either recombinant KIR3DL2 moleculesor surface-expressed KIR3DL2 molecules. For example, if a test antibodyreduces the binding of AZ158, 19H12, 2B12 or 12B11 to a KIR3DL2polypeptide or KIR3DL2-expressing cell in a binding assay, the antibodyis said to “compete” respectively with AZ158, 19H12, 2B12 or 12B11.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab−Ag], where[Ab−Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Coligan et al.,eds., Current Protocols in Immunology, Greene Publishing Assoc. and WleyInterscience, N.Y., (1992, 1993); and Muller, Meth. Enzymol. 92:589-601(1983), which references are entirely incorporated herein by reference.One standard method well-known in the art for determining the affinityof mAbs is the use of surface plasmon resonance (SPR) screening (such asby analysis with a BIAcore™ SPR analytical device).

A “determinant” designates a site of interaction or binding on apolypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody binds. A protein epitopemay comprise amino acid residues directly involved in the binding aswell as amino acid residues which are effectively blocked by thespecific antigen-binding antibody or peptide, i.e., amino acid residueswithin the “footprint” of the antibody. It is the simplest form orsmallest structural area on a complex antigen molecule that can combinewith, e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term “conformational” istherefore often used interchangeably with “structural”.

The term “immunogenic fragment” refers to any polypeptidic or peptidicfragment that is capable of eliciting an immune response such as (i) thegeneration of antibodies binding said fragment and/or binding any formof the molecule comprising said fragment, including the membrane-boundreceptor and mutants derived therefrom, or (ii) the stimulation of aT-cell response involving T cells reacting to the bi-molecular complexcomprising any MHC molecule and a peptide derived from said fragment.Alternatively, an immunogenic fragment also refers to any constructioncapable of eliciting an immune response as defined above, such as apeptidic fragment conjugated to a carrier protein by covalent coupling,or a chimeric recombinant polypeptide construct comprising said peptidicfragment in its amino acid sequence, and specifically includes cellstransfected with a cDNA of which the sequence comprises a portionencoding said fragment.

The terms “depleting”, “deplete” or “depletion”, with respect toKIR3DL2-expressing cells, mean a process, method, or compound that cankill, eliminate, lyse or induce such killing, elimination or lysis so asto negatively affect the number of KIR3DL2-expressing cells present in asample or in a subject.

The terms “immunoconjugate”, “antibody conjugate”, “antibody drugconjugate” and “ADC” are used interchangeably and refer to an antibodythat is conjugated to another moiety (e.g., any non-antibody moiety, atherapeutic agent or a label).

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “therapeutic agent” refers to anagent that has biological activity.

The terms “toxic agent”, “toxic moiety” and “cytotoxic agent” encompassany compound that can slow down, halt, or reverse the proliferation ofcells, decrease their activity in any detectable way, or directly orindirectly kill them. Cytotoxic agents can cause cell death primarily byinterfering directly with the cell's functioning, and include, but arenot limited to, alkylating agents, tumor necrosis factor inhibitors, DNAintercalators, microtubule inhibitors, kinase inhibitors, proteasomeinhibitors and topoisomerase inhibitors. A “toxic payload” as usedherein refers to a sufficient amount of cytotoxic agent which, whendelivered to a cell, result in cell death. Delivery of a toxic payloadmay be accomplished by administration of a sufficient amount ofimmunoconjugate comprising an antibody or antigen-binding fragment and acytotoxic agent. Delivery of a toxic payload may also be accomplished byadministration of a sufficient amount of an immunoconjugate comprising acytotoxic agent, wherein the immunoconjugate comprises a secondaryantibody or antigen-binding fragment thereof which recognizes and bindsan antibody or antigen-binding fragment.

The term “human-suitable”, with respect to an antibody, refers to anyantibody, derivatized antibody, or antibody fragment that can be safelyused in humans for, e.g., the therapeutic methods described herein.Human-suitable antibodies include all types of humanized, chimeric, orfully human antibodies, or any antibodies in which at least a portion ofthe antibody is derived from humans or otherwise modified so as to avoidthe immune response that is generally provoked when native non-humanantibodies are used.

A “humanized” or “human” antibody refers to an antibody in which theconstant and variable framework region of one or more humanimmunoglobulins is fused with the binding region, e.g., the CDR, of ananimal immunoglobulin. Such antibodies are designed to maintain thebinding specificity of the non-human antibody from which the bindingregions are derived, but to avoid an immune reaction against thenon-human antibody. Such antibodies can be obtained from transgenic miceor other animals that have been “engineered” to produce specific humanantibodies in response to antigenic challenge (see, e.g., Green et al.(1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Tayloret al. (1994) Int Immun 6:579, the entire teachings of which are hereinincorporated by reference). A fully human antibody also can beconstructed by genetic or chromosomal transfection methods, as well asphage display technology, all of which are known in the art (see, e.g.,McCafferty et al. (1990) Nature 348:552-553). Human antibodies may alsobe generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos.5,567,610 and 5,229,275, which are incorporated in their entirety byreference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen-binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc., or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ε and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, Md.).

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” iswell-understood in the art, and refers to a cell-mediated reaction inwhich non-specific cytotoxic cells that express Fc receptors (FcRs)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. Non-specific cytotoxic cells that mediate ADCCinclude natural killer (NK) cells, macrophages, monocytes, neutrophils,and eosinophils.

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “recombinant”, when used with reference, e.g., to a cell,nucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all.

As used herein, “T cells” refers to a sub-population of lymphocytes thatmature in the thymus, and which display, among other molecules, T cellreceptors on their surface. T cells can be identified by virtue ofcertain characteristics and biological properties, such as theexpression of specific surface antigens including TCR, CD4 or CD8,optionally CD4 and IL-23R, the ability of certain T cells to kill tumoror infected cells, the ability of certain T cells to activate othercells of the immune system, and the ability to release protein moleculescalled cytokines that stimulate or inhibit the immune response. Any ofthese characteristics and activities can be used to identify T cells,using methods well-known in the art.

“Prominently expressed”, when referring to a KIR3DL2 polypeptide, meansthat the KIR3DL2 polypeptide is expressed in a substantial number oftumor cells (e.g., PTCL cells, malignant or over-proliferating T or NKcells) taken from a given patient. While the definition of the term“prominently expressed” is not bound by a precise percentage value, inmost cases a receptor said to be “prominently expressed” will be presenton at least 30%, 40%, 50° %, 60%, 70%, 80%, or more of the PTCL cellstaken from a patient.

As used herein, an antibody that “binds” a polypeptide or epitopedesignates an antibody that binds said determinant with specificityand/or affinity.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithm”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje,G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M. Stockton Press, New York, 1991; and Carillo etal., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Computer programmethods for determining identity between two sequences include the GCGprogram package, including GAP (Devereux et al., Nucl. Acid. Res. 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison,Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215,403-410 (1990)). The BLASTX program is publicly available from theNational Center for Biotechnology Information (NCBI) and other sources(BLAST Manual, Altschul et al., NCB/NLM/NIH, Bethesda, Md. 20894;Altschul et al., supra). The well-known Smith-Waterman algorithm mayalso be used to determine identity.

Production of Antibodies

KIR3DL2 (CD158k) is a disulfide-linked homodimer of three-Ig domainmolecules of about 140 kD, described in Pende et al. (1996) J. Exp. Med.184: 505-518, the disclosure of which is incorporated herein byreference. Several allelic variants have been reported for KIR3DL2polypeptides; each of these are encompassed by the term KIR3DL2. Theamino acid sequence of the mature human KIR3DL2 (allele *002) is shownin SEQ ID NO: 1 below, corresponding to GenBank Accession No. AAB52520,in which the 21 amino acid residue leader sequence has been omitted:

(SEQ ID NO: 1) LMGGQDKPF  LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPLVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHRDGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV VSCPRAPQSG LEGVF. 

The cDNA of KIR3DL2 (allele *002) is shown in GenBank Accession No.U30272. The amino acid sequence of human KIR3DL2 allele *003 is shownbelow, corresponding to GenBank Accession No. AAB36593:

(SEQ ID NO: 2) MSLTVVSMAC VGFFLLQGAW PLMGGQDKPF LSARPSTVVP RGGHVALQCH YRRGFNNFML YKEDRSHVPI FHGRIFQESF IMGPVTPAHA GTYRCRGSRP HSLTGWSAPS NPVVIMVTGN HRKPSLLAHP GPLLKSGETV ILQCWSDVMF EHFFLHREGI SEDPSRLVGQ IHDGVSKANF SIGPLMPVLA GTYRCYGSVP HSPYQLSAPS DPLDIVITGL YEKPSLSAQP GPTVQAGENV TLSCSSWSSY DIYHLSREGE AHERRLRAVP KVNRTFQADF PLGPATHGGT YRCFGSFRAL PCVWSNSSDP LLVSVTGNPS SSWPSPTEPS SKSGICRHLH VLIGTSVVIF LFILLLFFLL YRWCSNKKNA AVMDQEPAGD RTVNRQDSDE QDPQEVTYAQ LDHCVFIQRK ISRPSQRPKT PLTDTSVYTE LPNAEPRSKV  VSCPRAPQSG LEGVF. 

Also encompassed are any nucleic acid or protein sequences sharing oneor more biological properties or functions with wild-type, full-lengthKIR3DL2, respectively, and sharing at least 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or higher nucleotide or amino acid identity.

Closely related KIR3DL1 (CD158e1) is a monomeric molecule of about 70kD, described in Colonna and Samaridis (1995) Science 268 (5209),405-408. The cDNA encoding a KIR3DL1 (CD158e2) polypeptide (allele*00101) is shown in GenBank Accession No. L41269; the encoded amino acidsequence is shown in GenBank Accession No. AAA69870. In one embodiment,a KIR3DL1 polypeptide referred to herein is allele *00101.

Examples of antibodies that bind human KIR3DL2 include antibody AZ158,antibody 19H12, antibody 2B12 and antibody 12B11. Further antibodies areprovided in the United States patent application publication numbers20150232556 and 20150291692, both of which are incorporated herein byreference. AZ158 binds human KIR3DL2 as well as human KIR3DL1 andKIR3DS1 polypeptides; 19H12, 2B12 and 12B11 bind selectively to KIR3DL2and do not bind KIR3DL1 (or KIR3DS1). While antibody AZ158 can be used,for example, as a therapeutic agent administered to an individual forthe elimination of a KIR3DL2-expressing target, e.g., by induction ofADCC and/or CDC, antibodies 12B11 and 19H12 will be advantageous overAZ158 for use in detection (e.g., in vitro assays) of KIR3DL2 expressionon the surface of tumor cells because 12B11 and 19H12 are both able todetect KIR3DL2-positive cells in detection assays; 12B11 is advantageousfor immunohistochemistry assays using frozen tissue sections, while19H12 is advantageous for flow cytometry detection. Each of 2B12, 19H12and 12B11 are also suitable for use as a therapeutic agent administeredto an individual for the elimination of KIR3DL2-expressing target cells.19H12 and 12B11 as well as other antibodies disclosed in the UnitedStates patent application publication number 20150291692 are capable ofbeing internalized into cells via KIR3DL2 and can be used advantageouslyas an antibody-drug conjugate. 2B12 and other antibodies disclosed inthe United States patent application publication 20150232556 do notinduce any KIR3DL2 internalization into tumor cells, thereby providingadvantageous use when effector cell-mediated activity is sought, e.g.,for depleting antibodies that induce ADCC.

In a specific embodiment, an antibody is provided that binds essentiallythe same epitope or determinant as any of monoclonal antibodies AZ158,19B12, 12B11 or 2B12; optionally the antibody comprises anantigen-binding region of antibody AZ158, 19B12, 12B11 or 2B12. In anyof the embodiments herein, antibody AZ158, 19B12, 12B11 or 2B12 can becharacterized by its amino acid sequence and/or the nucleic acidsequence encoding it. In one embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of AZ158, 19B12, 12B11 or 2B12.Also provided is a monoclonal antibody that comprises the heavy chainvariable region of AZ158, 19B12, 12B11 or 2B12. According to oneembodiment, the monoclonal antibody comprises the three CDRs of theheavy chain variable region of AZ158, 19B12, 12B11 or 2B12. Alsoprovided is a monoclonal antibody that further comprises the light chainvariable region of AZ158, 19B12, 12B11 or 2B12 or one, two or three ofthe CDRs of the light chain variable region of AZ158, 19B12, 12B11 or2B12. Optionally any one or more of said light or heavy chain CDRs maycontain one, two, three, four or five or more amino acid modifications(e.g., substitutions, insertions or deletions). Optionally, an antibodyis provided where any of the light and/or heavy chain variable regionscomprising part or all of an antigen-binding region of antibody AZ158,19B12, 12B11 or 2812 are fused to an immunoglobulin constant region ofthe human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

Antibody AZ158

AZ158 binds human KIR3DL2 as well as human KIR3DL1 polypeptides. AZ158can be characterized as having the heavy and light chain variableregions or heavy and light chain region CDRs of SEQ ID NOS: 8 and 10,respectively, of PCT patent publication no. WO2010/081890. The VH ofAZ158 is shown below, with CDRs 1, 2 and 3 underlined, respectively:

(SEQ ID NO: 3) QVQLKESGPG LVAPSQSLSI TCTVSGFSLT SFGVHWVRQP PGKGLEWLGV IWAGGSTNYN SALMSRLSIS KDNSKSQVFL KMNSLQNDDT AMYYCARGNS NHYVSSFYYF DYWGQGTTLT VSS.

The VL of AZ158 is shown below with CDRs 1, 2 and 3 underlined,respectively:

(SEQ ID NO: 4) DIQMTQSPSS LSASLGGKVT ITCKASQDIN KYIAWYQHKP GKGPRLLIHY TSTLQPGIPS RFSGSGSGRD YSFSISNLEPEDITTYYCLQ YDNLWTFGGG TKLEIK.

The anti-KIR3DL2 antibodies may include antibodies having variableregion or CDR sequences from such AZ158 antibodies (e.g., a heavy and/orlight chain variable region fused to a human constant region; a heavychain variable region fused to a human IgG1 heavy chain constantregion); alternatively, the anti-KIR3DL2 antibodies may be an antibodyother than the antibodies having variable region or CDR sequences froman AZ158 antibody.

Antibody 19H12

The amino acid sequence of the heavy chain variable region of antibody19H12 is listed below:

(SEQ ID NO: 5) QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARNG NFGYYFDYWGQGTTLTVSS. 

The amino acid sequence of the light chain variable region of antibody19H12 is listed below:

(SEQ ID NO: 6) DVLMTQTPLSLPVSLGDQASFSCRSSQNIVHSNGNTYLEWYLQKPGQSPSLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVP FTFGSGTKLEIK.

In one aspect, a purified polypeptide is provided which encodes anantibody, wherein the antibody comprises an HCDR1 region comprising anamino acid sequence GYTFTNFGMN as set forth in SEQ ID NO:9, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof (e.g., NFGMN (SEQ ID NO: 7), GYTFTN (SEQ ID NO: 8)), wherein oneor more of these amino acids may be substituted by a different aminoacid; an HCDR2 region comprising an amino acid sequence WINTYTGEPTYADDFas set forth in SEQ ID NO: 10, or a sequence of at least 4, 5, 6, 7, 8,9 or 10 contiguous amino acids thereof (e.g., WINTYTGE (SEQ ID NO: 11)),wherein one or more of these amino acids may be substituted by adifferent amino acid; an HCDR3 region comprising an amino acid sequenceNGNFGYYFDY as set forth in SEQ ID NO: 12, or a sequence of at least 4,5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or moreof these amino acids may be substituted by a different amino acid; anLCDR1 region comprising an amino acid sequence RSSQNIVHSNGNTYLE as setforth in SEQ ID NO: 13, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be substituted by a different amino acid; an LCDR2 region comprisingan amino acid sequence KVSNRFS as set forth in SEQ ID NO: 14, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; and/or an LCDR3 region comprising an amino acidsequence FQGSHVPFT as set forth in SEQ ID NO: 15, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein oneor more of these amino acids may be deleted or substituted by adifferent amino acid, or where the sequence may comprise an insertion ofone or more amino acids.

In another aspect, an antibody is provided that binds human KIR3DL2,comprising:

(a) the heavy chain variable region of SEQ ID NO: 5, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(b) the light chain variable region of SEQ ID NO: 6, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(c) the heavy chain variable region of SEQ ID NO: 5, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid, and the light chain variable region of SEQ ID NO: 6, whereinone or more of these amino acids may be substituted by a different aminoacid; and/or

(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 7-9, 10-11 and 12, respectively,wherein one, two, three or more amino acid residues of any CDR may besubstituted by a different amino acid; and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 13, 14 or 15, respectively, whereinone, two, three or more amino acid residues of any CDR may besubstituted by a different amino acid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 7, 8 or 9, 10 or 11 and 12,respectively, wherein one, two, three or more amino acid residues of anyCDR may be substituted by a different amino acid, and the light chainCDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown inSEQ ID NOS: 13, 14 or 15, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 5, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 6, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid.

Antibody 12B11

The amino acid sequence of the heavy chain variable region of antibody12B11 is listed below:

(SEQ ID NO: 16) QLVQSGPELKNPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCAHGPWL AYWGQGTLVTVS.

The amino acid sequence of the light chain variable region of antibody12B11 is listed below:

(SEQ ID NO: 17) DIKMTQSPSSMYASLGERVTITCKASQDINVYLSWFQQKPGKSPKTLIYRAIRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYDELPYTFGG GTKLEIE.

In one aspect, a purified polypeptide is provided which encodes anantibody, wherein the antibody comprises: an HCDR1 region comprising anamino acid sequence GYTFTNYGMN as set forth in SEQ ID NO: 20, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof (e.g., NYGMN (SEQ ID NO: 18), GYTFTN (SEQ ID NO: 19)), whereinone or more of these amino acids may be substituted by a different aminoacid; an HCDR2 region comprising an amino acid sequenceWINTYTGEPTYADDFKG as set forth in SEQ ID NO: 21, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof (e.g.,WINTYTGEPT (SEQ ID NO: 22)), wherein one or more of these amino acidsmay be substituted by a different amino acid; an HCDR3 region comprisingan amino acid sequence GPWLAY as set forth in SEQ ID NO: 23, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; an LCDR1 region comprising an amino acidsequence KASQDINVYLS as set forth in SEQ ID NO: 24, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein oneor more of these amino acids may be substituted by a different aminoacid; an LCDR2 region comprising an amino acid sequence RAIRLVD as setforth in SEQ ID NO: 25, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be substituted by a different amino acid; and/or an LCDR3 regioncomprising an amino acid sequence LQYDELPYT as set forth in SEQ ID NO:26, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may be deletedor substituted by a different amino acid.

In another aspect, an antibody is provided that binds human KIR3DL2,comprising:

(a) the heavy chain variable region of SEQ ID NO: 16, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(b) the light chain variable region of SEQ ID NO: 17, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(c) the heavy chain variable region of SEQ ID NO: 16, wherein one ormore amino acid residues may be substituted by a different amino acid,and the light chain variable region of SEQ ID NO: 17, wherein one, two,three or more of these amino acids may be substituted by a differentamino acid; and/or

(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 18, 19 or 20, 21 or 22 and 23,respectively, wherein one, two, three or more amino acid residues of anyCDR may be substituted by a different amino acid; and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 24, 25 and 26, wherein one, two, threeor more amino acid residues of any CDR may be substituted by a differentamino acid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 18, 19 or 20, 21 or 22 and 23,respectively, wherein one or more amino acid residues of any CDR may besubstituted by a different amino acid, and the light chain CDR 1, 2 and3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOS: 24,25 and 26, wherein one, two, three or more amino acid residues of anyCDR may be substituted by a different amino acid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 16, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 17, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid.

Antibody 2B12

The amino acid sequence of the heavy chain variable region of antibody2B12 is listed below (Kabat definition CDRs underlined):

(SEQ ID NO: 32) Q I Q L V Q S G P E L K K P G E T V R I S C K A SG Y T F T T A G M Q W V Q K T P G K G L K W I G WI N S H S G V P K Y A E D F K G R F A F S L E T SA S T A Y L Q I S T L K N E D T A T Y F C A R G GD E G V M D Y W G Q G T S V T V S.

The amino acid sequence of the light chain variable region of antibody2B12 is listed below (CDRs underlined):

(SEQ ID NO: 33) D I V M T Q S H K F M S T S L G D R V S F T C K AS Q D V S T A V A W Y Q Q K P G Q S P K L L I Y WT S T R H T G V P D R F T G S G S G T D Y T L T IS S V Q A E D L A L Y Y C Q Q H Y S T P W T F G G G T K L E I K.

In one aspect, a purified polypeptide is provided which encodes anantibody, wherein the antibody comprises: an HCDR1 region comprising anamino acid sequence GYTFTTAGMQ as set forth in SEQ ID NO: 36, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof (e.g., GYTFTT (SEQ ID NO: 34) or TAGMQ (SEQ ID NO: 35)), whereinone or more of these amino acids may be substituted by a different aminoacid; an HCDR2 region comprising an amino acid sequence WINSHSGVPKYAEDFKas set forth in SEQ ID NO: 37, or a sequence of at least 4, 5, 6, 7, 8,9 or 10 contiguous amino acids thereof (e.g., WINSHSGVP (SEQ ID NO:38)), wherein one or more of these amino acids may be substituted by adifferent amino acid; an HCDR3 region comprising an amino acid sequenceGGDEGVMDYW as set forth in SEQ ID NO: 39, or a sequence of at least 4,5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or moreof these amino acids may be substituted by a different amino acid; anLCDR1 region comprising an amino acid sequence KASQDVSTAVA as set forthin SEQ ID NO: 40, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be substituted by a different amino acid; an LCDR2 region comprisingan amino acid sequence WTSTRHT as set forth in SEQ ID NO: 41, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; and/or an LCDR3 region comprising an amino acidsequence QQHYSTPVVT as set forth in SEQ ID NO: 42, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein oneor more of these amino acids may be deleted or substituted by adifferent amino acid.

In another aspect, an antibody is provided that binds human KIR3DL2,comprising:

(a) the heavy chain variable region of SEQ ID NO: 32, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(b) the light chain variable region of SEQ ID NO: 33, wherein one, two,three or more amino acid residues may be substituted by a differentamino acid; and/or

(c) the heavy chain variable region of SEQ ID NO: 32, wherein one ormore amino acid residues may be substituted by a different amino acid,and the light chain variable region of SEQ ID NO: 33, wherein one, two,three or more of these amino acids may be substituted by a differentamino acid; and/or

(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 34, 35 or 36, 37 or 38 and 39,respectively, wherein one, two, three or more amino acid residues of anyCDR may be substituted by a different amino acid; and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 40, 41 and 42, wherein one, two, threeor more amino acid residues of any CDR may be substituted by a differentamino acid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 34, 35 or 36, 37 or 38 and 39,respectively, wherein one or more amino acid residues of any CDR may besubstituted by a different amino acid, and the light chain CDR 1, 2 and3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOS: 40,41 and 42, wherein one, two, three or more amino acid residues of anyCDR may be substituted by a different amino acid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 32, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 33, wherein one, two, three or more amino acidresidues may be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, an antibody is provided that competes for KIR3DL2binding with a monoclonal antibody of (a) to (h), for any of the aboveantibodies.

Antibody Epitopes

While it will be appreciated that any suitable antibody can be used, inone aspect the antibodies that are used bind substantially the sameepitope as antibody 19H12 or 12B11. In another embodiment, theantibodies at least partially overlap, or include at least one residuein the segment corresponding to residues 1-192, residues 1-98, orresidues 99-192 of the KIR3DL2 polypeptide of SEQ ID NO: 1 (or asubsequence thereof). In one embodiment, all key residues of the epitopeare in a segment corresponding to residues 1-192, residues 1-98 orresidues 99-192 of the KIR3DL2 polypeptide of SEQ ID NO: 1. In oneembodiment, the antibodies bind an epitope comprising 1, 2, 3, 4, 5, 6,7 or more residues in the segment corresponding to residues 1-192, 1-98or 99-192 of the KIR3DL2 polypeptide of SEQ ID NO: 1. Preferably theresidues bound by the antibody are present on the surface of the KIR3DL2polypeptide.

Optionally, the antibodies bind an epitope comprising residue P179and/or residue S181 of SEQ ID NO: 1. Optionally, the antibodies bind toan epitope comprising 1, 2, 3, 4, 5, 6 or 7 or more residues selectedfrom the group consisting of N99, H100, E130, H131, F132, V178, P179,H180, S181, P182, Y183 and/or residue Q184 of SEQ ID NO: 1.

The Examples section herein describes the testing of a series of mutanthuman KIR3DL2 polypeptides. Binding of anti-KIR3DL2 antibody to cellstransfected with the KIR3DL2 mutants was measured and compared to theability of anti-KIR3DL2 antibody to bind wild-type KIR3DL2 polypeptide(SEQ ID NO: 1). A reduction in binding between an anti-KIR3DL2 antibodyand a mutant KIR3DL2 polypeptide as used herein means that there is areduction in binding affinity (e.g., as measured by known methods suchFACS testing of cells expressing a particular mutant, or Biacore testingof binding to mutant polypeptides) and/or a reduction in the totalbinding capacity of the anti-KIR3DL2 antibody (e.g., as evidenced by adecrease in Bmax in a plot of anti-KIR3DL2 antibody concentration versuspolypeptide concentration). A significant reduction in binding indicatesthat the mutated residue is directly involved in binding to theanti-KIR3DL2 antibody or is in close proximity to the binding proteinwhen the anti-KIR3DL2 antibody is bound to KIR3DL2. An antibody epitopemay thus include such residue and may include additional residuesspatially adjacent to such residue.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an anti-KIR3DL2 antibody and amutant KIR3DL2 polypeptide is reduced by greater than 40%, greater than50%, greater than 55%, greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90% or greater than 95% relative to binding between the antibody and awild-type KIR3DL2 polypeptide (e.g., the polypeptide shown in SEQ ID NO:1). In certain embodiments, binding is reduced below detectable limits.In some embodiments, a significant reduction in binding is evidencedwhen binding of an anti-KIR3DL2 antibody to a mutant KIR3DL2 polypeptideis less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15% or10%) of the binding observed between the anti-KIR3DL2 antibody and awild-type KIR3DL2 polypeptide (e.g., the extracellular domain shown inSEQ ID NO: 1). Such binding measurements can be made using a variety ofbinding assays known in the art. A specific example of one such assay isdescribed in the Examples section.

In some embodiments, anti-KIR3DL2 antibodies are provided that exhibitsignificantly lower binding for a mutant KIR3DL2 polypeptide in which aresidue in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO: 1) issubstituted. In the shorthand notation used here, the format is: Wildtype residue: Position in polypeptide: Mutant residue, with thenumbering of the residues as indicated in SEQ ID NO: 1.

Optionally, the antibodies have reduced binding to a KIR3DL2 polypeptidehaving a substitution at residues N99, H100, E130, H131, F132, V178,P179, H180, S181, P182, Y183 and/or Q184 of SEQ ID NO: 1.

In some embodiments, an anti-KIR3DL2 antibody binds a wild-type KIR3DL2polypeptide having a sequence of SEQ ID NO: 1 but has decreased bindingto a mutant KIR3DL2 polypeptide having any one or more (e.g., 1, 2, 3 or4) of the following mutations: P179T and/or S181T (with reference to SEQID NO: 1). In one embodiment, binding to the mutant KIR3DL2 issignificantly reduced compared to binding to the wild-type KIR3DL2. Insome embodiments, anti-KIR3DL2 antibodies are provided that exhibitsignificantly lower binding for a mutant KIR3DL2 polypeptide in which aresidue in a segment corresponding to residues 1-98, residues 99-292, orresidues 99-192 (or a subsequence thereof) in a wild-type KIR3DL2polypeptide (e.g., SEQ ID NO: 1) is substituted with a different aminoacid.

In one aspect, an antibody can compete with monoclonal antibody AZ158,19H12, 2B12 or 12B11 and recognize binding to, or have immunospecificityfor substantially or essentially the same, or the same, epitope or“epitopic site” on a KIR3DL2 molecule as, monoclonal antibody AZ158,19H12, 2B12 or 12B11. In other embodiments, the monoclonal antibodyconsists of, or is a derivative or fragment of, antibody AZ158, 19H12,2B12 or 12B11.

It will be appreciated that, while antibodies may bind to the sameepitope as antibody AZ158, 19H12, 2B12 or 12B11, suitable antibodies canrecognize and be raised against any part of the KIR3DL2 polypeptide solong as the antibody binds KIR3DL2 and has the desired functionality.For example, any fragment of KIR3DL2, e.g., human KIR3DL2, or anycombination of KIR3DL2 fragments, can be used as immunogens to raiseantibodies, and the antibodies can recognize epitopes at any locationwithin the KIR3DL2 polypeptide, so long as they can do so onKIR3DL2-expressing NK cells as described herein. In an embodiment, therecognized epitopes are present on the cell surface, i.e., they areaccessible to antibodies present outside of the cell. Optionally, theepitope is the epitope specifically recognized by antibody AZ158, 19H12,2B12 or 12B11. Further, antibodies recognizing distinct epitopes withinKIR3DL2 can be used in combination, e.g., to bind to KIR3DL2polypeptides with maximum efficacy and breadth among differentindividuals.

The antibodies may be produced by a variety of techniques known in theart. Typically, they are produced by immunization of a non-human animal,optionally a mouse, with an immunogen comprising a KIR3DL2 polypeptide,optionally a human KIR3DL2 polypeptide. The KIR3DL2 polypeptide maycomprise the full-length sequence of a human KIR3DL2 polypeptide, or afragment or derivative thereof, typically an immunogenic fragment, i.e.,a portion of the polypeptide comprising an epitope exposed on thesurface of cells expressing a KIR3DL2 polypeptide, optionally theepitope recognized by the AZ158, 19H12, 2B12 or 12B11 antibody. Suchfragments typically contain at least about 7 consecutive amino acids ofthe mature polypeptide sequence, or at least about 10 consecutive aminoacids thereof. Fragments typically are essentially derived from theextracellular domain of the receptor. In one embodiment, the immunogencomprises a wild-type human KIR3DL2 polypeptide in a lipid membrane,typically at the surface of a cell. In one embodiment, the immunogencomprises intact cells, particularly intact human cells, optionallytreated or lysed. In another embodiment, the polypeptide is arecombinant KIR3DL2 polypeptide.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well-known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). The immunogen is suspended ordissolved in a buffer, optionally with an adjuvant, such as complete orincomplete Freund's adjuvant. Methods for determining the amount ofimmunogen, types of buffers and amounts of adjuvant are well-known tothose of skill in the art and are not limiting in any way. Theseparameters may be different for different immunogens, but are easilyelucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well-known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with an adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe used as long as they result in the production of B cells expressingan antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunizednon-human animal and the antibodies present therein are isolated bywell-known techniques. The serum may be affinity purified using any ofthe immunogens set forth above linked to a solid support so as to obtainantibodies that react with KIR3DL2 polypeptides.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For exemplary monoclonal antibodies, the next step is the isolation ofsplenocytes from the immunized non-human mammal and the subsequentfusion of those splenocytes with an immortalized cell in order to forman antibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsulethrough a nylon mesh of a cell strainer into an appropriate buffer so asto produce a single-cell suspension. The cells are washed, centrifugedand resuspended in a buffer that lyses any red blood cells. The solutionis again centrifuged and remaining lymphocytes in the pellet are finallyresuspended in fresh buffer.

Once isolated and present in single-cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Murine myeloma lines include, but arenot limited to, those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,U.S.A., and X63 Ag8653 and SP-2 cells available from the American TypeCulture Collection, Rockville, Md. U.S.A. The fusion is effected usingpolyethylene glycol or the like. The resulting hybridomas are then grownin selective media that contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages can be from littermates of the non-human mammal used toisolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for a sufficienttime for colony formation and antibody production. This is usuallybetween about 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodiesthat specifically bind to KIR3DL2 polypeptide gene products, optionallythe epitope specifically recognized by antibody AZ158, 19H12, 2B12 or12B11. The assay is typically a colorimetric ELISA-type assay, althoughany assay may be employed that can be adapted to the wells that thehybridomas are grown in. Other assays include radioimmunoassays orfluorescence-activated cell sorting. The wells positive for the desiredantibody production are examined to determine if one or more distinctcolonies are present. If more than one colony is present, the cells maybe re-cloned and grown to ensure that only a single cell has given riseto the colony producing the desired antibody. Typically, the antibodieswill also be tested for the ability to bind to KIR3DL2 polypeptides,e.g., KIR3DL2-expressing cells.

Hybridomas that are confirmed to produce a monoclonal antibody can begrown in larger amounts in an appropriate medium, such as DMEM orRPMI-1640. Alternatively, the hybridoma cells can be grown in vivo asascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing the monoclonal antibody (or the ascites fluid)is separated from the cells and the monoclonal antibody present thereinis purified. Purification is typically achieved by gel electrophoresis,dialysis, chromatography using protein A or protein G Sepharose, or ananti-mouse Ig linked to a solid support such as agarose or Sepharosebeads (all described, for example, in the Antibody PurificationHandbook, Biosciences, publication No. 18-1037-46, Edition AC, thedisclosure of which is hereby incorporated by reference). The boundantibody is typically eluted from protein A/protein G columns by usinglow pH buffers (glycine or acetate buffers of pH 3.0 or less) withimmediate neutralization of antibody-containing fractions. Thesefractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in Ward et al., Nature,341 (1989), p. 544, the entire disclosure of which is hereinincorporated by reference.

The identification of one or more antibodies that bind(s) to KIR3DL2,particularly substantially or essentially the same epitope as monoclonalantibody AZ158, 19H12, 2B12 or 12B11, can be readily determined usingany one of a variety of immunological screening assays in which antibodycompetition can be assessed. Many such assays are routinely practicedand are well-known in the art (see, e.g., U.S. Pat. No. 5,660,827,issued Aug. 26, 1997, which is specifically incorporated herein byreference). It will be understood that actually determining the epitopeto which an antibody described herein binds is not in any way requiredto identify an antibody that binds to the same or substantially the sameepitope as the monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (AZ158,19H12, 2B12 or 12B11, for example) and test antibodies are admixed (orpre-adsorbed) and applied to a sample containing KIR3DL2 polypeptides.Protocols based upon Western blotting and the use of BIACORE analysisare suitable for use in such competition studies.

In certain embodiments, one pre-mixes the control antibodies (AZ158,19H12, 2B12 or 12B11, for example) with varying amounts of the testantibodies (e.g., about 1:10 or about 1:100) for a period of time priorto applying to the KIR3DL2 antigen sample. In other embodiments, thecontrol and varying amounts of test antibodies can simply be admixedduring exposure to the KIR3DL2 antigen sample. As long as one candistinguish bound from free antibodies (e.g., by using separation orwashing techniques to eliminate unbound antibodies) and AZ158, 19H12,2B12 or 12B11 from the test antibodies (e.g., by using species-specificor isotype-specific secondary antibodies or by specifically labelingAZ158, 19H12, 2B12 or 12B11 with a detectable label) one can determineif the test antibodies reduce the binding of AZ158, 19H12, 2B12 or 12B11to the antigens, indicating that the test antibody recognizessubstantially the same epitope as AZ158, 19H12, 2B12 or 12B11. Thebinding of the (labeled) control antibodies in the absence of acompletely irrelevant antibody can serve as the control high value. Thecontrol low value can be obtained by incubating the labeled (AZ158,19H12, 2B12 or 12B11) antibodies with unlabeled antibodies of exactlythe same type (AZ158, 19H12, 2B12 or 12B11), where competition wouldoccur and reduce binding of the labeled antibodies. In a test assay, asignificant reduction in labeled antibody reactivity in the presence ofa test antibody is indicative of a test antibody that recognizessubstantially the same epitope, i.e., one that “cross-reacts” orcompetes with the labeled (AZ158, 19H12, 2B12 or 12B11) antibody. Anytest antibody that reduces the binding of AZ158, 19H12, 2B12 or 12B11 toKIR3DL2 antigens by at least about 50%, such as at least about 60%, ormore preferably at least about 80% or 90% (e.g., about 65-100%), at anyratio of AZ158, 19H12, 2B12 or 12B11:test antibody between about 1:10and about 1:100 is considered to be an antibody that binds tosubstantially the same epitope or determinant as AZ158, 19H12, 2B12 or12B11. For example such test antibody will reduce the binding of AZ158,19H12, 2B12 or 12B11 to the KIR3DL2 antigen by at least about 90% (e.g.,about 95%).

Competition can also be assessed by, for example, a flow cytometry test.In such a test, cells bearing a given KIR3DL2 polypeptide can beincubated first with AZ158, 19H12, 2B12 or 12B11, for example, and thenwith the test antibody labeled with a fluorochrome or biotin. Theantibody is said to compete with AZ158, 19H12, 2B12 or 12B11 if thebinding obtained upon preincubation with a saturating amount of AZ158,19H12, 2B12 or 12B11 is about 80%, about 50%, about 40% or less (e.g.,about 30%, 20% or 10%) of the binding (as measured by means offluorescence) obtained by the antibody without pre-incubation withAZ158, 19H12, 2B12 or 12B11. Alternatively, an antibody is said tocompete with AZ158, 19H12, 2B12 or 12B11 if the binding obtained with alabeled AZ158, 19H12, 2B12 or 12B11 antibody (by a fluorochrome orbiotin) on cells preincubated with a saturating amount of test antibodyis about 80%, about 50%, about 40%, or less (e.g., about 30%, 20% or10%) of the binding obtained without preincubation with the testantibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which a KIR3DL2antigen is immobilized may also be employed. The surface in the simplecompetition assay is for example a BIACORE chip (or other media suitablefor surface plasmon resonance analysis). The control antibody (e.g.,AZ158, 19H12, 2B12 or 12B11) is then brought into contact with thesurface at a KIR3DL2-saturating concentration and the KIR3DL2 andsurface binding of the control antibody is measured. This binding of thecontrol antibody is compared with the binding of the control antibody tothe KIR3DL2-containing surface in the absence of test antibody. In atest assay, a significant reduction in binding of the KIR3DL2-containingsurface by the control antibody in the presence of a test antibodyindicates that the test antibody recognizes substantially the sameepitope as the control antibody such that the test antibody“cross-reacts” with the control antibody. Any test antibody that reducesthe binding of control (such as AZ158, 19H12, 2B12 or 12B11) antibody toa KIR3DL2 antigen by at least about 30% or more, or about 40%, can beconsidered to be an antibody that binds to substantially the sameepitope or determinant as a control (e.g., AZ158, 19H12, 2B12 or 12B11).For example, such a test antibody will reduce the binding of the controlantibody (e.g., AZ158, 19H12, 2B12 or 12B11) to the KIR3DL2 antigen byat least about 50% (e.g., at least about 60%, at least about 70%, ormore). It will be appreciated that the order of control and testantibodies can be reversed: that is, the control antibody can be firstbound to the surface and the test antibody is brought into contact withthe surface thereafter in a competition assay. For example, the antibodyhaving higher affinity for the KIR3DL2 antigen is bound to the surfacefirst, as it will be expected that the decrease in binding seen for thesecond antibody (assuming the antibodies are cross-reacting) will be ofgreater magnitude. Further examples of such assays are provided in,e.g., Saunal (1995) J. Immunol. Methods 183: 33-41, the disclosure ofwhich is incorporated herein by reference.

Determination of whether an antibody binds within an epitope region canbe carried out in ways known to the person skilled in the art. As oneexample of such mapping/characterization methods, an epitope region foran anti-KIR3DL2 antibody may be determined by epitope “footprinting”using chemical modification of the exposed amines/carboxyls in theKIR3DL2 protein. One specific example of such a footprinting techniqueis the use of HXMS (hydrogen-deuterium exchange detected by massspectrometry) wherein a hydrogen/deuterium exchange of receptor andligand protein amide protons, binding, and back exchange occurs, whereinthe backbone amide groups participating in protein binding are protectedfrom back exchange and therefore will remain deuterated. Relevantregions can be identified at this point by peptic proteolysis, fastmicrobore high-performance liquid chromatography separation, and/orelectrospray ionization mass spectrometry. See, e.g., Ehring, H.,Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999); Engen, J. R.and Smith, D. L. (Anal. Chem. 73, 256A-265A (2001). Another example of asuitable epitope identification technique is nuclear magnetic resonanceepitope mapping (NMR), where typically the position of the signals intwo-dimensional NMR spectra of the free antigen and the antigencomplexed with the antigen-binding peptide, such as an antibody, arecompared. The antigen is typically selectively isotopically labeled with15N so that only signals corresponding to the antigen and no signalsfrom the antigen-binding peptide are seen in the NMR spectrum. Antigensignals originating from amino acids involved in the interaction withthe antigen-binding peptide will typically shift position in thespectrum of the complex compared to the spectrum of the free antigen,and the amino acids involved in the binding can be identified that way.See, e.g., Ernst Schering Res. Found. Workshop, 44:149-67 (2004); Huanget al., Journal of Molecular Biology, 281(1):61-67 (1998); and Saito andPatterson, Methods, 1996 June, 9(3):516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downard, J. Mass. Spectrom, 2000 April,35(4):493-503 and Kiselar and Downard, Anal. Chem., 1999 May 1,71(9):1792-801. Protease digestion techniques can also be useful in thecontext of epitope mapping and identification. Antigenicdeterminant-relevant regions/sequences can be determined by proteasedigestion, e.g., by using trypsin in a ratio of about 1:50 to KIR3DL2 oro/n digestion at pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe anti-KIR3DL2 binder can subsequently be identified by comparison ofsamples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by, e.g., trypsin (therebyrevealing a footprint for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the KIR3DL2 polypeptide that is notsurface exposed and, accordingly, most likely not relevant in terms ofimmunogenicity/antigenicity. See, e.g., Manca, Ann 1st Super Sanita,1991, 27:15-9 for a discussion of similar techniques.

Site-directed mutagenesis is another technique useful for elucidation ofa binding epitope. For example, in “alanine-scanning”, each residuewithin a protein segment is replaced with an alanine residue, and theconsequences for binding affinity are measured. If the mutation leads toa significant reduction in binding affinity, it is most likely involvedin binding. Monoclonal antibodies specific for structural epitopes(i.e., antibodies which do not bind the unfolded protein) can be used toverify that the alanine replacement does not influence the overall foldof the protein. See, e.g., Clackson and Wells, Science 1995,267:383-386, and Wells, Proc Natl Acad Sci USA 1996, 93:1-6.

Electron microscopy can also be used for epitope “footprinting”. Forexample, Wang et al., Nature 1992, 355:275-278 used coordinatedapplication of cryoelectron microscopy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include surfaceplasmon resonance (SPR, BIACORE) and reflectometric interferencespectroscopy (RifS). See, e.g., Fagerstam et al., Journal of MolecularRecognition 1990, 3:208-14; Nice et al., J. Chromatogr. 1993,646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998, 37:3308-3311;Kröger et al., Biosensors and Bioelectronics 2002, 17:937-944.

It should also be noted that an antibody binding the same orsubstantially the same epitope as an antibody can be identified in oneor more of the exemplary competition assays described herein.

Once antibodies are identified that are capable of binding KIR3DL2and/or having other desired properties, they will also typically beassessed, using standard methods including those described herein, fortheir ability to bind to other polypeptides, including unrelatedpolypeptides. Ideally, the antibodies only bind with substantialaffinity to KIR3DL2, e.g., human KIR3DL2, and do not bind at asignificant level to unrelated polypeptides. However, it will beappreciated that, as long as the affinity for KIR3DL2 is substantiallygreater (e.g., 5×, 10×, 50×, 100×, 500×, 1000×, 10,000×, or more) thanit is for other, unrelated polypeptides, the antibodies are suitable foruse in the present methods.

The binding of the antibodies to KIR3DL2-expressing cells can also beassessed in non-human primates, e.g., cynomolgus monkeys, or othermammals such as mice. Provided is an antibody, as well as fragments andderivatives thereof, wherein said antibody, fragment or derivativespecifically binds KIR3DL2, and which furthermore binds KIR3DL2 fromnon-human primates, e.g., cynomolgus monkeys.

Upon immunization and production of antibodies in a vertebrate or cell,particular selection steps may be performed to isolate antibodies asclaimed. In this regard, in a specific embodiment, the disclosure alsorelates to methods of producing such antibodies, comprising: (a)immunizing a non-human mammal with an immunogen comprising a KIR3DL2polypeptide; (b) preparing antibodies from said immunized animal; and(c) selecting antibodies from step (b) that are capable of bindingKIR3DL2.

In one aspect of any of the embodiments, the antibodies preparedaccording to the present methods are monoclonal antibodies. In anotheraspect, the non-human animal used to produce antibodies is a mammal,such as a rodent, bovine, porcine, fowl, horse, rabbit, goat, or sheep.

According to an alternate embodiment, the DNA encoding an antibody thatbinds an epitope present on KIR3DL2 polypeptides is isolated from thehybridoma and placed in an appropriate expression vector fortransfection into an appropriate host. The host is then used for therecombinant production of the antibody, or variants thereof, such as ahumanized version of that monoclonal antibody, active fragments of theantibody, chimeric antibodies comprising the antigen recognition portionof the antibody, or versions comprising a detectable moiety.

DNA encoding a monoclonal antibody, e.g., antibody 19H12, 2B12 or 12B11,can be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of murineantibodies). Once isolated, the DNA can be placed into expressionvectors, which are then transfected into host cells, such as E. colicells, simian COS cells, Chinese hamster ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells. Asdescribed elsewhere in the present specification, such DNA sequences canbe modified for any of a large number of purposes, e.g., for humanizingantibodies, producing fragments or derivatives, or for modifying thesequence of the antibody, e.g., in the antigen-binding site in order tooptimize the binding specificity of the antibody.

Recombinant expression in bacteria of DNA encoding the antibody iswell-known in the art (see, for example, Skerra et al., Curr. Opinion inImmunol., 5, pp. 256 (1993), and Pluckthun, Immunol. 130, p. 151(1992)).

Once an antigen-binding compound is obtained it may be assessed for itsability to induce ADCC or CDC towards, inhibit the activity and/orproliferation of and/or cause the elimination of KIR3DL2-expressingtarget cells. Assessing the antigen-binding compound's ability to induceADCC or CDC (complement-dependent cytotoxicity) or generally lead to theelimination or inhibition of activity of KIR3DL2-expressing target cellscan be carried out at any suitable stage of the method. This assessmentcan be useful at one or more of the various steps involved in theidentification, production and/or development of an antibody (or othercompound) destined for therapeutic use. For example, activity may beassessed in the context of a screening method to identify candidateantigen-binding compounds, or in methods where an antigen-bindingcompound is selected and made human suitable (e.g., made chimeric orhumanized in the case of an antibody), where a cell expressing theantigen-binding compound (e.g., a host cell expressing a recombinantantigen-binding compound) has been obtained and is assessed for itsability to produce functional antibodies (or other compounds), and/orwhere a quantity of antigen-binding compound has been produced and is tobe assessed for activity (e.g., to test batches or lots of product).Generally the antigen-binding compound will be known to specificallybind to a KIR3DL2 polypeptide. The step may involve testing a plurality(e.g., a very large number using high-throughput screening methods or asmaller number) of antigen-binding compounds.

Testing CDC and ADCC can be carried out by various assays includingthose known in the art and those described in the experimental examplesherein. Testing ADCC typically involves assessing cell-mediatedcytotoxicity in which a KIR3DL2-expressing target cell (e.g., a PTCLcell or other KIR3DL2-expressing cell) with bound anti-KIR3DL2 antibodyis recognized by an effector cell bearing Fc receptors, without theinvolvement of complement. A cell which does not express a KIR3DL2antigen can optionally be used as a control. Activation of NK cellcytotoxicity is assessed by measuring an increase in cytokine production(e.g., IFN-γ production) or cytotoxicity markers (e.g., CD107mobilization). In one embodiment, the antibody will induce an increasein cytokine production, expression of cytotoxicity markers, or targetcell lysis of at least 20%, 50%, 80%, 100%, 200% or 500% in the presenceof target cells, compared to a control antibody (e.g., an antibody notbinding to KIR3DL2, a KIR3DL2 antibody having murine constant regions).In another example, lysis of target cells is detected, e.g., in achromium release assay; for example the antibody will induce lysis of atleast 10%, 20%, 30%, 40% or 50% of the target cells.

Fragments and derivatives of antibodies (which are encompassed by theterm “antibody” or “antibodies” as used in this application, unlessotherwise stated or clearly contradicted by context) can be produced bytechniques that are known in the art. “Fragments” comprise a portion ofthe intact antibody, generally the antigen-binding site or variableregion. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)2, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single-chain polypeptide”),including without limitation (1) single-chain Fv molecules, (2)single-chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety and (3)single-chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety; andmultispecific antibodies formed from antibody fragments. Included, interalia, are a nanobody, domain antibody, single domain antibody or a“dAb”.

In certain embodiments, the DNA of a hybridoma producing an antibody canbe modified prior to insertion into an expression vector, for example,by substituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous non-human sequences (e.g.,Morrison et al., PNAS pp. 6851 (1984)), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of theoriginal antibody. Typically, such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody.

Thus, according to another embodiment, the antibody is humanized.“Humanized” forms of antibodies are specific chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)2, or other antigen-binding subsequences of antibodies) whichcontain minimal sequences derived from the murine immunoglobulin. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementarity-determining region(CDR) of the recipient are replaced by residues from a CDR of theoriginal antibody (donor antibody) while maintaining the desiredspecificity, affinity, and capacity of the original antibody.

In some instances, Fv framework residues of the human immunoglobulin maybe replaced by corresponding non-human residues. Furthermore, humanizedantibodies can comprise residues that are not found in either therecipient antibody or in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof the original antibody and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al., Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et al., Science, 239,pp. 1534; and U.S. Pat. No. 4,816,567, the entire disclosures of whichare herein incorporated by reference. Methods for humanizing theantibodies are well-known in the art.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of the mouse is then accepted as the humanframework (FR) for the humanized antibody (Sims et al., J. Immunol. 151,pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, 1987, pp. 901). Anothermethod uses a particular framework from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework can be used for several different humanized antibodies(Carter et al., PNAS 89, pp. 4285 (1992); Presta et al., J. Immunol.,151, p. 2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for KIR3DL2 receptors and other favorable biologicalproperties. To achieve this goal, according to one method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen(s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse (Abgenix, Fremont, Calif.) as the mouse for immunization. AXenoMouse is a murine host that has had its immunoglobulin genesreplaced by functional human immunoglobulin genes. Thus, antibodiesproduced by this mouse or in hybridomas made from the B cells of thismouse are already humanized. The XenoMouse is described in U.S. Pat. No.6,162,963, which is herein incorporated in its entirety by reference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al., Nature 362:255 (1993)), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

A KIR3DL2 binding compound, e.g., an anti-KIR3DL2 antibody, may befurther bound to a second moiety, wherein the antibody is capable ofdelivering the second moiety to a KIR3DL2-expressing cell. Optionallythe second moiety is a therapeutic agent, a toxic agent, and/or adetectable agent.

While antibodies in underivatized or unmodified form, particularly ofthe IgG1 or IgG3 type, are expected to inhibit the proliferation of theoverproliferating cells or be cytotoxic towards overproliferating cellssuch as in those from a PTCL patient, e.g., by directing ADCC and/or CDCtoward KIR3DL2-expressing PTCL cells, it is also possible to preparederivatized antibody immunoconjugates that are cytotoxic. In oneembodiment, once the KIR3DL2-specific antibodies are isolated andoptionally otherwise modified (e.g., humanized), they will bederivatized to make them toxic to cells. In this way, administration ofthe antibody to PTCL patients will lead to the relatively specificbinding of the antibody to overproliferating cells, thereby directlykilling or inhibiting the cells underlying the disorder.

Any of a large number of toxic moieties or strategies can be used toproduce such antibodies. In certain embodiments, the antibodies will bedirectly derivatized with radioisotopes or other toxic compounds.Examples of toxic agents used in immunoconjugates in development includetaxanes, anthracyclines, camptothecins, epothilones, mitomycins,combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids,calicheamycins, duocarmycins, tubulysins, dolastatins, auristatins,enediynes, pyrrolobenzodiazepines, ethylenimines, radioisotopes,therapeutic proteins and peptides, and toxins or fragments thereof. Anytype of moiety with a cytotoxic or cytoinhibitory effect can be used inconjunction with the present antibodies to inhibit or kill specific NKreceptor-expressing cells, including radioisotopes, toxic proteins, andtoxic small molecules, such as drugs, toxins, immunomodulators,hormones, hormone antagonists, enzymes, oligonucleotides, enzymeinhibitors, therapeutic radionuclides, angiogenesis inhibitors,chemotherapeutic drugs, vinca alkaloids, epidophyllotoxins,antimetabolites, alkylating agents, antibiotics, antimitotics,antiangiogenic and apoptotic agents, particularly doxorubicin,methotrexate, camptothecins, nitrogen mustards, gemcitabine, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, platinum coordination complexes, Pseudomonasexotoxin, ricin, 5-fluorouridine, ribonuclease (RNase), DNase I,staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, and others (see, e.g., Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995); Goodman and Gilman's ThePharmacological Basis of Therapeutics (McGraw-Hill, 2001); Pastan et al.(1986) Cell 47:641; Goldenberg (1994) Cancer Journal for Clinicians44:43; and U.S. Pat. No. 6,077,499, the entire disclosures of which areherein incorporated by reference).

In one embodiment, the antibody will be derivatized with a radioactiveisotope, such as 1-131. Any of a number of suitable radioactive isotopescan be used, including, but not limited to, Indium-111, Lutetium-171,Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64, Copper-67,Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32, Phosphorus-33,Scandium-47, Silver-111, Gallium-67, Praseodymium-142, Samarium-153,Terbium-161, Dysprosium-166, Holmium-166, Rhenium-186, Rhenium-188,Rhenium-189, Lead-212, Radium-223, Actinium-225, Iron-59, Selenium-75,Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105, Palladium-109,Praseodymium-143, Promethium-149, Erbium-169, Iridium-194, Gold-198,Gold-199, and Lead-211. The radionuclide may have a decay energy in therange of 20 to 6,000 keV, optionally in the ranges of 60 to 200 keV foran Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keVfor an alpha emitter. Also provided are radionuclides that substantiallydecay with generation of alpha particles.

In view of the ability of the anti-KIR3DL2 antibodies to induce ADCC andCDC, the antibodies can also be made with modifications that increasetheir ability to bind Fc receptors which can affect effector functionssuch as antibody-dependent cytotoxicity, mast cell degranulation, andphagocytosis, as well as immunomodulatory signals such as regulation oflymphocyte proliferation and antibody secretion. Typical modificationsinclude modified human IgG1 constant regions comprising at least oneamino acid modification (e.g., substitutions, deletions, insertions),and/or altered types of glycosylation, e.g., hypofucosylation. Suchmodifications can affect interaction with Fc receptors: FcγRI (CD64),FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) andFcγRIII (CD16) are activating (i.e., immune system-enhancing) receptorswhile FcγRIIB (CD32B) is an inhibiting (i.e., immune system-dampening)receptor. A modification may, for example, increase binding of the Fcdomain to FcγRIIIa on effector (e.g., NK) cells.

Anti-KIR3DL2 antibodies may comprise an Fc domain (or portion thereof)of human IgG1 or IgG3 isotype, optionally modified. Residues 230-341(Kabat EU) are the Fc CH2 region. Residues 342-447 (Kabat EU) are the FcCH3 region. Anti-KIR3DL2 antibodies may comprise a variant Fc regionhaving one or more amino acid modifications (e.g., substitutions,deletions, insertions) in one or more portions, which modificationsincrease the affinity and avidity of the variant Fc region for an FcγR(including activating and inhibitory FcγRs). In some embodiments, saidone or more amino acid modifications increase the affinity of thevariant Fc region for FcγRIIIA and/or FcγRIIA. In another embodiment,the variant Fc region further specifically binds FcγRIIB with a loweraffinity than does the Fc region of the comparable parent antibody(i.e., an antibody having the same amino acid sequence as the antibodyexcept for the one or more amino acid modifications in the Fc region).For example, one or both of the histidine residues at amino acidpositions 310 and 435 may be substituted, for example by lysine,alanine, glycine, valine, leucine, isoleucine, proline, methionine,tryptophan, phenylalanine, serine or threonine (see, e.g., PCTpublication no. WO 2007/080277); such substituted constant regionsprovide decreased binding to the inhibitory FcγRIIB without decreasingbinding to the activatory FcγRIIIA. In some embodiments, suchmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA and also enhance the affinity of the variant Fcregion for FcγyRIIB relative to the parent antibody. In otherembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but do notalter the affinity of the variant Fc regions for FcγRIIB relative to theFc region of the parent antibody. In another embodiment, said one ormore amino acid modifications enhance the affinity of the variant Fcregion for FcγRIIIA and FcγRIIA but reduce the affinity for FcγRIIBrelative to the parent antibody. Increased affinity and/or avidityresults in detectable binding to the FcγR or FcγR-related activity incells that express low levels of the FcγR when binding activity of theparent molecule (without the modified Fc region) cannot be detected inthe cells.

The affinities and binding properties of the molecules for an FcγR canbe determined using in vitro assays (biochemical or immunological-basedassays) known in the art for determining antibody-antigen or Fc-FcγRinteractions, i.e., specific binding of an antigen to an antibody orspecific binding of an Fc region to an FcγR, respectively, including butnot limited to ELISA assay, surface plasmon resonance assay, andimmunoprecipitation assay.

In some embodiments, the molecules comprising a variant Fc regioncomprise at least one amino acid modification (for example, possessing1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH3domain of the Fc region. In other embodiments, the molecules comprisinga variant Fc region comprise at least one amino acid modification (forexample, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acidmodifications) in the CH2 domain of the Fc region, which is defined asextending from amino acids 231-341. In some embodiments, the moleculescomprise at least two amino acid modifications (for example, possessing2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein atleast one such modification is in the CH3 region and at least one suchmodification is in the CH2 region. Amino acid modifications may be madefor example in the hinge region. In a particular embodiment, theinvention encompasses amino acid modification in the CH1 domain of theFc region, which is defined as extending from amino acids 216-230.

Any combination of Fc modifications can be made, for example anycombination of different modifications disclosed in U.S. Pat. Nos.7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008;7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505; and 6,737,056; inPCT Publication Nos. WO2011/109400; WO 2008/105886; WO 2008/002933; WO2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474;WO 04/1032269; WO 00/42072; WO 06/088494; WO 07/024249; WO 05/047327; WO04/099249; and WO 04/063351; and in Presta, L. G. et al. (2002) Biochem.Soc. Trans. 30(4):487-490; Shields, R. L. et al. (2002) J. Biol. Chem.277(30):26733-26740; and Shields, R. L. et al. (2001) J. Biol. Chem.276(9):6591-6604).

Anti-KIR3DL2 antibodies may comprise a variant Fc region, wherein thevariant Fc region comprises at least one amino acid modification (forexample, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acidmodifications) relative to a wild-type Fc region, such that the moleculehas an enhanced effector function relative to a molecule comprising awild-type Fc region, optionally wherein the variant Fc region comprisesa substitution at any one or more of positions 221, 239, 243, 247, 255,256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 308, 309,310, 311, 312, 316, 320, 322, 326, 329, 330, 332, 331, 332, 333, 334,335, 337, 338, 339, 340, 359, 360, 370, 373, 376, 378, 392, 396, 399,402, 404, 416, 419, 421, 430, 434, 435, 437, 438 and/or 439.

Anti-KIR3DL2 antibodies may comprise a variant Fc region, wherein thevariant Fc region comprises at least one amino acid modification (forexample, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acidmodifications) relative to a wild-type Fc region, such that the moleculehas an enhanced effector function relative to a molecule comprising awild-type Fc region, optionally wherein the variant Fc region comprisesa substitution at any one or more of positions 329, 298, 330, 332, 333and/or 334 (e.g., S239D, S298A, A330L, I332E, E333A and/or K334Asubstitutions).

In one embodiment, antibodies having variant or wild-type Fc regions mayhave altered glycosylation patterns that increase Fc receptor bindingability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies to thereby produce anantibody with altered glycosylation. See, for example, Shields, R. L. etal. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat.Biotech. 17:176-1; European Patent No. EP 1,176,195; and PCT PublicationNos. WO 06/133148, WO 03/035835, and WO 99/54342, each of which isincorporated herein by reference in its entirety.

Generally, such antibodies with altered glycosylation are“glyco-optimized” such that the antibody has a particular N-glycanstructure that produces certain desirable properties, including, but notlimited to, enhanced ADCC and effector cell receptor-binding activitywhen compared to non-modified antibodies or antibodies having anaturally occurring constant region and produced by murine myeloma NSOand Chinese Hamster Ovary (CHO) cells (Chu and Robinson, Current OpinionBiotechnol. 2001, 12: 180-7), HEK293T-expressed antibodies as producedherein in the Examples section, or other mammalian host cell linescommonly used to produce recombinant therapeutic antibodies.

Monoclonal antibodies produced in mammalian host cells contain anN-linked glycosylation site at Asn297 of each heavy chain. Glycans onantibodies are typically complex biantennary structures with very low orno bisecting N-acetylglucosamine (bisecting GlcNAc) and high levels ofcore fucosylation. Glycan termini contain very low or no terminal sialicacid and variable amounts of galactose. For a review of effects ofglycosylation on antibody function, see, e.g., Wright & Morrison, TrendsBiotechnol. 15:26-31 (1997). Considerable work shows that changes to thesugar composition of the antibody glycan structure can alter Fc effectorfunctions. The important carbohydrate structures contributing toantibody activity are believed to be the fucose residues attached viaalpha-1,6 linkage to the innermost N-acetylglucosamine (GlacNAc)residues of the Fc region N-linked oligosaccharides (Shields et al.,2002).

FcγR binding requires the presence of oligosaccharides covalentlyattached at the conserved Asn297 in the Fc region of human IgGI, IgG2 orIgG3 type. Non-fucosylated oligosaccharide structures have recently beenassociated with dramatically increased in vitro ADCC activity. “Asn297”means amino acid asparagine located at about position 297 in the Fcregion; based on minor sequence variations of antibodies, Asn297 canalso be located some amino acids (usually not more than +3 amino acids)upstream or downstream.

Historically, antibodies produced in CHO cells contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 (rat myeloma) and Lecl3cell lines (a lectin mutant of the CHO line which has a deficientGDP-mannose 4,6-dehydratase leading to the deficiency of GDP-fucose orGDP sugar intermediates that are the substrate ofalpha6-fucosyltransferase) have been reported to produce antibodies with78 to 98% non-fucosylated species. In other examples, RNA interference(RNAi) or knock-out techniques can be employed to engineer cells toeither decrease the FUT8 mRNA transcript levels or knock out geneexpression entirely, and such antibodies have been reported to containup to 70% non-fucosylated glycan.

An antibody that binds to KIR3DL2 may be glycosylated with a sugar chainat Asn297. In one embodiment, an antibody will comprise a constantregion comprising at least one amino acid alteration in the Fc regionthat improves antibody binding to FcγRIIIa and/or ADCC.

In one aspect, the antibodies are hypofucosylated in their constantregion. Such antibodies may comprise an amino acid alteration or may notcomprise an amino acid alteration but be produced or treated underconditions so as to yield such hypofucosylation. In one aspect, anantibody composition comprises a chimeric, human or humanized antibodydescribed herein, wherein at least 20, 30, 40, 50, 60, 75, 85, 90, 95%or substantially all of the antibody species in the composition have aconstant region comprising a core carbohydrate structure (e.g., complex,hybrid and high mannose structures) which lacks fucose. In oneembodiment, an antibody composition is provided which is free ofantibodies comprising a core carbohydrate structure having fucose. Thecore carbohydrate will preferably be a sugar chain at Asn297.

In one embodiment, an antibody composition, e.g., a compositioncomprising antibodies which bind to KIR3DL2, is glycosylated with asugar chain at Asn297, wherein the antibodies are partially fucosylated.Partially fucosylated antibodies are characterized in that theproportion of anti-KIR3DL2 antibodies in the composition that lackfucose within the sugar chain at Asn297 is between 20% and 90%, between20% and 80%, between 20% and 50%, 55%, 60%, 70% or 75%, between 35% and50%, 55%, 60%, 70% or 75%, or between 45% and 50%, 55%, 60%, 70% or 75%.Optionally the antibody is of human IgGI or IgG3 type.

The sugar chain can further show any characteristics (e.g., presence andproportion of complex, hybrid and high mannose structures), includingthe characteristics of N-linked glycans attached to Asn297 of anantibody from a human cell, or of an antibody recombinantly expressed ina rodent cell, murine cell (e.g., CHO cell) or avian cell.

In one embodiment, the antibody is expressed in a cell that is lackingin a fucosyltransferase enzyme such that the cell line produces proteinslacking fucose in their core carbohydrates. For example, the cell linesMs704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in the Ms704,Ms705, and Ms709 cell lines lack fucose on their core carbohydrates.These cell lines were created by the targeted disruption of the FUT8gene in CHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22, the disclosures of which areincorporated herein by reference). Other examples have included the useof antisense suppression, double-stranded RNA (dsRNA) interference,hairpin RNA (hpRNA) interference or intron-containing hairpin RNA(ihpRNA) interference to functionally disrupt the FUT8 gene. In oneembodiment, the antibody is expressed in a cell line with a functionallydisrupted FUT8 gene, which encodes a fucosyl transferase, such thatantibodies expressed in such a cell line exhibit hypofucosylation byreducing or eliminating the alpha 1,6 bond-related enzyme.

In one embodiment, the antibody is expressed in cell lines engineered toexpress glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyl-transferase III (GnTHI)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures, which results in increased ADCC activity ofthe antibodies (PCT Publication WO 99/54342 by Umana et al. and Umana etal. (1999) Nat. Biotech. 17:176-180, the disclosures of which areincorporated herein by reference).

In another embodiment, the antibody is expressed and the fucosylresidue(s) is cleaved using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino et al. (1975) Biochem. 14:5516-5523). In other examples, acell line producing an antibody can be treated with a glycosylationinhibitor; Zhou et al. (2008) Biotech. and Bioengin. 99:652-665described treatment of CHO cells with the alpha-mannosidase I inhibitorkifunensine, resulting in the production of antibodies withnon-fucosylated oligomannose-type N-glucans.

In one embodiment, the antibody is expressed in a cell line whichnaturally has a low enzyme activity for adding fucosyl to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). Other examples of cell lines include a variantCHO cell line, Led 3 cells, with reduced ability to attach fucosyl toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (WO 03/035835 (Presta et al); andShields, R. X. et al. (2002) J. Biol. Chem. 277:26733-26740, thedisclosures of which are incorporated herein by reference). In anotherembodiment, the antibody is expressed in an avian cell, e.g., an EBx®cell (Vivalis, France), which naturally yields antibodies with lowfucose content, e.g., WO2008/142124. Hypofucosylated glycans can also beproduced in cell lines of plant origin, e.g., WO 07/084926A2 (BiolexInc.), WO 08/006554 (Greenovation Biotech GmbH), the disclosures ofwhich are incorporated herein by reference.

Antibody Formulations

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins such as human serum albumin,buffer substances such as phosphates, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, andzinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. The antibodies may be employed in a method of modulating,e.g., inhibiting, the activity of KIR3DL2-expressing cells in a patient.This method comprises the step of contacting said composition with saidpatient. Such method will be useful for both prophylaxis and therapeuticpurposes.

For use in administration to a patient, the composition will beformulated for administration to the patient. The compositions may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The administration routes used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques.

Sterile injectable forms of the compositions may be aqueous oroleaginous suspensions. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives, are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms, may also be used forthe purposes of formulation.

Several monoclonal antibodies have been shown to be efficient inclinical situations, such as Rituxan™ (Rituximab), Herceptin™(Trastuzumab) or Xolair™ (Omalizumab), and similar administrationregimens (i.e., formulations, doses and/or administration protocols) maybe used with the antibodies. For example, an antibody present in apharmaceutical composition can be supplied at a concentration of 10mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. Theproduct is formulated for IV administration in 9.0 mg/mL sodiumchloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80,and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplarysuitable dosage range for an antibody in a pharmaceutical compositionmay be between about 1 mg/m² and 500 mg/m². However, it will beappreciated that these schedules are exemplary and that an optimalschedule and regimen can be adapted taking into account the affinity andtolerability of the particular antibody in the pharmaceuticalcomposition that must be determined in clinical trials. A pharmaceuticalcomposition for injection (e.g., intramuscular, i.v.) could be preparedto contain sterile buffered water (e.g., 1 ml for intramuscular), andfrom about 1 ng to about 100 mg, e.g., about 50 ng to about 30 mg ormore, e.g., about 5 mg to about 25 mg, of an anti-KIR3DL2 antibody.

According to another embodiment, the antibody compositions may furthercomprise another therapeutic agent, including agents normally utilizedfor the particular therapeutic purpose for which the antibody is beingadministered, notably for the treatment of a PTCL.

The additional therapeutic agent will normally be present in thecomposition in amounts typically used for that agent in a monotherapyfor the particular disease or condition being treated. Such therapeuticagents include, but are not limited to, anti-inflammation agents,steroids, immune system suppressors, antibiotics, antivirals and otherantibodies and fragments thereof.

Diagnosis and Treatment of Malignancies

Provided are methods useful in the diagnosis, prognosis and monitoringof a peripheral T cell lymphoma in an individual. In one embodiment, themethods comprise determining the level of expression of a KIR3DL2nucleic acid or polypeptide in a biological sample from a patient, e.g.,in tumor cells found in a biological sample. In one embodiment, themethods comprise determining the level of expression of a KIR3DL2nucleic acid or polypeptide in a biological sample and comparing thelevel to a reference level (e.g., a value, weak cell surface staining,etc.) corresponding to a healthy individual(s). A determination that abiological sample expresses a KIR3DL2 nucleic acid or polypeptide at alevel that is increased compared to the reference level indicates thatthe patient has a peripheral T cell lymphoma. Optionally, detecting aKIR3DL2 polypeptide in a biological sample comprises detecting a KIR3DL2polypeptide expressed on the surface of a lymphocyte.

In one embodiment, the methods comprise: (a) determining whether anindividual has a peripheral T cell lymphoma; and (b) if the individualhas a peripheral T cell lymphoma, determining whether the individual hasperipheral T cell lymphoma cells that express a KIR3DL2 polypeptide.

Also provided is a method for the assessment of the development level ofa PTCL (staging disease) permitting the evaluation of the proportion(e.g., percentage) of malignant PTCL cells present within a certain bodycompartment of a patient. According to this method, cells from abiological sample collected from said body compartment are brought intocontact with an anti-KIR3DL2 antibody and tumor cells (e.g., theproportion of cells) expressing a KIR3DL2 polypeptide at their surfaceare measured. The cells may be, for example CD4+ cells or CD4-CD8+cells. A finding that tumor cells express, or predominantly express,KIR3DL2 indicates that the PTCL is an aggressive or advanced PTCL (e.g.,stage IV, or more generally beyond stage II).

Also provided is a method for PTCL diagnosis, comprising bringing cellsfrom a biological sample from an individual into contact with ananti-KIR3DL2 antibody and measuring the proportion (e.g., percentage) ofT cells expressing a KIR3DL2 polypeptide at their surface, and comparingsuch proportion to the average proportion (e.g., percentage) of T cellsexpressing a KIR3DL2 polypeptide at their surface observed in non-PTCLhumans (e.g., in healthy humans), wherein a PTCL-positive diagnosis ismade when said measured proportion is significantly higher than saidaverage proportion.

Further provided are therapeutic methods for treating individuals havinga PTCL, susceptible to a PTCL or having experienced a PTCL, wherein thetreatment involves administering anti-KIR3DL2 antibodies, anti-KIR3DL2antibody compositions, and/or related compositions to an individualhaving or susceptible to PTCL. In one embodiment, the PTCL is anaggressive or advanced PTCL (e.g., stage IV, or more generally beyondstage II). In one embodiment, the PTCL is a non-cutaneous PTCL. In oneembodiment, the PTCL is an aggressive T cell lymphoma. In oneembodiment, the patient has relapsing or refractory disease. In oneembodiment, the patient has a poor prognosis for disease progression(e.g., poor prognosis for survival) or has a poor prognosis for responseto a therapy, e.g., antibody therapy, anti-CD30 antibody therapy,chemotherapy, etc.

In one embodiment, the PTCL is an aggressive T-cell neoplasm. In oneembodiment, the PTCL is aggressive non-cutaneous PTCL. In oneembodiment, the PTCL is an aggressive cutaneous PTCL, optionally aprimary cutaneous CD4+ small/medium T cell lymphoma or a primary CD8+small/medium T cell lymphoma. PTCL and PTCL-NOS may be specified to bediseases other than cutaneous T cell lymphomas, Sézary Syndrome andmycosis fungoides, which are considered distinct pathologies.

In one embodiment, the PTCL is a nodal (e.g., primarily or predominantlynodal) PTCL. Predominantly nodal PTCLs include, inter alia, peripheralT-cell lymphomas, not otherwise specified (PTCL-NOS), anaplastic largecell lymphomas (ALCL) and angioimmunoblastic T-cell lymphomas (AITL).For example a PTCL may be an aggressive, non-cutaneous, predominantlynodal PCTL (the disease may additionally have extra-nodal presentation).

In one embodiment, the PTCL is an extranodal (e.g., primarilyextranodal) PTCL. For example a PTCL may be an aggressive,non-cutaneous, extranodal PCTL.

In one embodiment, the PTCL is an adult T cell leukemia or lymphoma(ATL), e.g., an HTLV+ ATL.

In one embodiment, the PTCL is an extranodal NK/T cell lymphoma, nasaltype. In one embodiment, the PTCL is an enteropathy-associated T celllymphoma.

In one embodiment, the PTCL is a hepatosplenic T cell lymphoma,optionally a hepatosplenic αβ T cell lymphoma, optionally ahepatosplenic γδ T cell lymphoma.

In one embodiment, the PTCL is an anaplastic large cell lymphoma (ALCL),optionally an ALK+ ALCL, optionally an ALK− ALCL. ALK+ ALCL generallyenjoys favorable prognostics using conventional therapy (93% 5-yearsurvival) but ALK− ALCL has poor prognostics (37%). ALCL is generallycharacterized by uniform CD30 surface expression. Anti-KIR3DL2antibodies can therefore be used in combination with anti-CD30antibodies (e.g., Adcetris™ (brentuximab vedotin, Seattle Genetics,Inc.)) for the treatment of ALCL. ALCL is generally also CD4⁺, althoughwith occasional CD4⁻ CD8⁺ cases. Anti-KIR3DL2 antibodies can thereforebe used in combination with anti-CD4 antibodies to treat ALCL. In oneembodiment, the PTCL is an angioimmunoblastic T-cell lymphoma (AITL),optionally a cutaneous AITL, optionally a primary cutaneous CD4⁺small/medium T cell lymphoma or a primary CD8⁺ small/medium T celllymphoma, optionally a non-cutaneous AITL.

In one embodiment, the PTCL is an intestinal lymphoma, e.g., anintestinal ALCL.

In one embodiment, the PTCL is a T-cell prolymphocytic leukemia.

In one embodiment, a PTCL is a PTCL-NOS (peripheral T-cell lymphoma, nototherwise specified). PTCL-NOS, also referred to as PCTL-U orPTCL-unspecified, are aggressive lymphomas, mainly of nodal type, butextranodal involvement is common. The majority of nodal cases are CD4⁺and CD8⁻, and CD30 can be expressed in large cell variants. Mostpatients with PTCL-NOS present with nodal involvement; however, a numberof extranodal sites may also be involved (e.g., liver, bone marrow,gastrointestinal, skin). Studies generally report 5-year overallsurvival of approximately 30%-35% using standard chemotherapy. In thepast, a number of definite entities corresponding to recognizablesubtypes of T-cell neoplasm, such as Lennert lymphoma, T-zone lymphoma,pleomorphic T-cell lymphoma and T-immunoblastic lymphoma, have beendescribed, but evidence that these correspond to distinctiveclinicopathological entities is still lacking. For this reason therecent World Health Organization (WHO) classification of thehematopoietic and lymphoid neoplasms has collected these under thesingle broad category of PTCL-NOS/U. PTCL-NOS may therefore be specifiedto exclude certain distinctive clinicopathological entities such asT-cell prolymphocytic leukemia, ATL/adult T cell leukemia, extranodalNK/T cell leukemia (nasal type), EATL/enteropathy-type T cell lymphoma,hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T celllymphoma, ALCL/anaplastic large-cell lymphoma, and/orAITL/angioimmunoblastic T cell lymphoma. Anti-KIR3DL2 antibodies can beused in combination with anti-CD4 antibodies to treat PTCL-NOS.Anti-KIR3DL2 antibodies can be used in combination with anti-CD30antibodies to treat PTCL-NOS that are CD30⁺.

PTCL diagnosis criteria can be those of standard medical guidelines, forexample, according to the World Health Organization (WHO) classificationsystem (see, e.g., World Health Organization. WHO Classification ofTumours of Haematopoietic and Lymphoid Tissues, 4^(th) ed., Lyon,France: IARC Press, 2008). See also, e.g., Foss et al. (2011) Blood117:6756-6767, the disclosures of which are incorporated herein byreference.

In one exemplary aspect, a method is provided of reducing progression ofPTCL in a mammalian host (e.g., a human patient) having a detectablelevel of cancer cells, comprising administering an anti-KIR3DL2antibody, an anti-KIR3DL2 antibody composition, or a related composition(e.g., a nucleic acid encoding an anti-KIR3DL2 antibody) in an amountsufficient to detectably reduce the progression of the hematologicalmalignancies in the host.

In one exemplary aspect, a method is provided of treating PTCL in anindividual having a poor disease prognosis and/or who has relapsed, isresistant or is not responsive to therapy with a first therapeuticagent.

Disease or cancer diagnosis and progression can be defined by standardcriteria for the particular type of disease. PTCL (e.g., PTCL-NOS) istypically based on examination of peripheral blood or tissue biopsy forhistological features supplemented by detailed immunohistochemistry,flow cytometry, cytogenetics and molecular genetics. Examination mayinclude, for example, full blood count and differential, tests of renaland hepatic function, lactate dehydrogenase (LDH), Beta2 microglobulin,albumin, serum calcium, uric acid, bone marrow biopsy, chest X-ray andcomputerized tomography (CT) scan of chest, abdomen and pelvis.Progression is optionally determined by assessing the selective clonalexpansion of initiated cells. Methods for detecting cancers and cancerprogression can be achieved by any suitable technique, several examplesof which are known in the art. Examples of suitable techniques includePCR and RT-PCR (e.g., of cancer cell-associated genes or “markers”),biopsy, imaging techniques, karyotyping and other chromosomal analysis,immunoassay/immunocytochemical detection techniques, histological and/orhistopathology assays, cell kinetic studies and cell cycle analysis,flow cytometry, and physical examination techniques (e.g., for physicalsymptoms).

In one embodiment, diagnosing or assessing PTCL (e.g., PTCL-NOS)comprises chromosomal analysis. Cases of PTCL-NOS often showheterogeneous, variable morphology, with losses having been reported in3q, 6q, 9p, 10q, 12q and/or 5q, and recurrent chromosomal gains,including in 8q, 9p and/or 19q. One CGH study in PTCL-NOS has shownfrequent gains of 7q22-31, 1q, 3p, 5p and 8q24qter and losses of 6q22-24and 10p13pter, and cases with complex karyotypes had poor diseaseprognosis.

In one embodiment, diagnosing or assessing PTCL comprises biomarkeranalysis. In one embodiment, a patient having a poor disease prognosisis identified by biomarker analysis wherein the presence or absence(e.g., level of) a nucleic acid or protein is detected in a biologicalsample from the patient (e.g., in tumor cells from a patient). A rangeof biomarkers are known in PTCL, including, e.g., p53, Ki-67, BCL-2,BCL-XL, CD26, EBV, MDR, CCND2, CCR4, NK-Kb, CCR3, CXCR3, PRDM1, andALK-1.

In one embodiment, diagnosing or assessing PTCL comprises detectingchemokine receptors CXCR3 and/or CCR4 (e.g., detecting the presence orabsence of CXCR3 and/or CCR4 nucleic acid or proteins, or the levels ofCXCR3 and/or CCR4 nucleic acid or proteins). CXCR3 and CCR4 have beenfound respectively in 63% and 34% of PTCL-NOS (Percy et al., Int. ClassDiseases for Oncol. (ICD-O-3), 3^(rd) ed., Geneva, Switzerland, WorldHealth Organization (2000)). In one embodiment, a determination that apatient has a PTCL (e.g., PTCL cells) that is CXCR3-positive andCCR4-negative indicates that the patient has a poor disease prognosis.

Delivering anti-KIR3DL2 antibodies to a subject (either by directadministration or expression from a nucleic acid therein, such as from apox viral gene transfer vector comprising anti-KIR3DL2 antibody-encodingnucleic acid sequence(s)) and practicing the other methods herein can beused to reduce, treat, prevent, or otherwise ameliorate any suitableaspect of cancer progression (notably PTCL progression). The methodsherein can be particularly useful in the reduction and/or ameliorationof tumor growth (e.g., percentage (tumor cells compared to healthy Tcells), number of tumor cells in circulation) and any parameter orsymptom associated therewith (e.g., biomarkers). Methods that reduce,prevent, or otherwise ameliorate such aspects of cancer progression,independently and collectively, are advantageous features.

In another aspect, a method is provided of reducing the risk of cancerprogression, reducing the risk of further cancer progression in a cellpopulation that has undergone initiation, and/or providing a therapeuticregimen for reducing cancer progression in a human patient, whichcomprises administering to the patient one or more first treatments(e.g., induction therapy, such as a chemotherapeutic agent or anantibody) in an amount and regimen sufficient to achieve a response(partial or complete response), and then administering an amount of ananti-KIR3DL2 antibody or related composition (or applying a combinationadministration method) to the patient.

In a further aspect, a method is provided of promoting remission of aPTCL in a mammalian host, such as a human patient, comprisingadministering a composition comprising an anti-KIR3DL2 antibody to thehost, so as to promote PTCL remission in the host.

In an even further aspect, a method is provided for reducing the risk ofdeveloping a PTCL, reducing the time to onset of a cancerous condition,and/or reducing the severity of a PTCL diagnosed in the early stages,comprising administering to a host a prophylactically effective amountof an anti-KIR3DL2 antibody or related composition so as to achieve thedesired physiological effect(s).

In a further aspect, a method is provided of increasing the likelihoodof survival over a relevant period of a human patient diagnosed withPTCL. In another aspect, a method is provided for improving the qualityof life of a PTCL patient, comprising administering to the patient acomposition in an amount effective to improve the quality of lifethereof. In a further aspect, methods described herein can be applied tosignificantly reduce the number of PTCL cells in a vertebrate host, suchthat, for example, the total number of PTCL cells is reduced. In arelated sense, a method is provided for killing (e.g., either directlyor indirectly causing the death of) PTCL cells in a vertebrate, such asa human cancer patient.

According to another embodiment, the antibody compositions may be usedin combined treatments with one or more other therapeutic agents,including agents normally utilized for the particular therapeuticpurpose for which the antibody is being administered, notably for thetreatment of a PTCL. The additional therapeutic agent will normally beadministered in amounts and treatment regimens typically used for thatagent in a monotherapy for the particular disease or condition beingtreated.

For example, a second therapeutic agent may include one or morechemotherapeutic drugs, tumor vaccines, antibodies that bind totumor-specific antigens on tumor cells (e.g., anti-CD30 antibodies),antibodies that induce ADCC toward tumor cells, antibodies thatpotentiate immune responses, etc.). Further anti-cancer agents includealkylating agents, cytotoxic antibiotics such as topoisomerase Iinhibitors, topoisomerase II inhibitors, plant derivatives, RNA/DNAantimetabolites, and antimitotic agents. Examples may include cisplatin(CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, taxol, gemcitabine,navelbine, transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, or any analog or derivative variant of the foregoing.

Drugs currently used or tested for the treatment of PTCL include, interalia, chemotherapeutic agents such as CHOP (cyclophosphamide,doxorubicin, vincristine and prednisone), anthracyclines, antifolates,conjugates such as anti-CD25 fused to Pseudomonas toxin, IL-2 targetingdomain fused with diphtheria toxin, anti-CD30 antibody conjugated toauristatins (monomethylauristatin-E), HDAC inhibitors, lenalidomide,monoclonal antibodies such as anti-CD52, anti-VEGF (bevacizumab),anti-CD30 (adcetris), anti-CCR4, anti-CD4 (e.g., zanolimumab) andanti-CD2, nucleoside analogues such as cladribine, clofarabine,fludarabine, gemcitabine, nelarabine and pentostatin, proteosomeinhibitors such as bortezomib and signaling inhibitors such as selectiveinhibitors of protein kinase C (e.g., enzastaurin) or syk inhibitors(e.g., R788).

In the treatment methods, the KIR3DL2-binding compound and the secondtherapeutic agent can be administered separately, together orsequentially, or in a cocktail. In some embodiments, the KIR3DL2-bindingcompound is administered prior to the administration of the secondtherapeutic agent. For example, the KIR3DL2-binding compound can beadministered approximately 0 to 30 days prior to the administration ofthe second therapeutic agent. In some embodiments, a KIR3DL2-bindingcompound is administered from about 30 minutes to about 2 weeks, fromabout 30 minutes to about 1 week, from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 6hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day,or from about 1 to 5 days prior to the administration of the secondtherapeutic agent. In some embodiments, a KIR3DL2-binding compound isadministered concurrently with the administration of the therapeuticagents. In some embodiments, a KIR3DL2-binding compound is administeredafter the administration of the second therapeutic agent. For example, aKIR3DL2-binding compound can be administered approximately 0 to 30 daysafter the administration of the second therapeutic agent. In someembodiments, a KIR3DL2-binding compound is administered from about 30minutes to about 2 weeks, from about 30 minutes to about 1 week, fromabout 1 hour to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 6 hours, from about 6 hours to about 8 hours,from about 8 hours to 1 day, or from about 1 to 5 days after theadministration of the second therapeutic agent.

EXAMPLES Example 1—Generation of KIR3DL2-Selective Antibodies

Antibodies which bind KIR3DL2 but not closely-related KIR3DL1 weregenerated by immunizing mice with recombinant KIR3DL2-Fc fusion protein.Supernatants (SN) of the growing hybridomas were tested by flowcytometry on Sézary Syndrome cell lines (HUT78, COU-L) andHEK-293T/KIR3DL2 Domain 0-eGFP. Potentially interesting hybridomasselected from the initial screening were cloned by limiting dilutiontechniques in 96-well plates. The secondary screen involved selection ofhybridomas of interest by testing supernatants of the subclones by flowcytometry on HUT78, COU-L, HEK-293T/KIR3DL1 Domain 0-eGFP andHEK-293T/KIR3DL2 Domain 0-eGFP. Positive subclones were injected intomice to produce ascitis and antibodies of interest were purified beforebeing tested in a Biacore assay using rec KIR3DL2 chips, followed byvarious assay formats based on binding to human KIR3DL2-expressingcells.

Sequences of the variable domains of the heavy (VH) and light (VL)chains of antibodies were amplified by PCR from the cDNA of eachantibody. Sequences amplified were run on agarose gel then purifiedusing the Qiagen Gel Extraction kit. VH and VL sequences were thensubcloned into the Lonza expression vectors (Double-Gene Vectors) usingthe In-Fusion system (Clontech) according to the manufacturer'sinstructions. After sequencing, vectors containing the VH and VLsequences were prepared as maxiprep using the Promega PureYield™ PlasmidMaxiprep System. Vectors were then used for HEK-293T cell transfectionusing Invitrogen's Lipofectamine 2000 according to the manufacturer'sinstructions. Antibodies generated included, inter alia, 19H12 and12B11.

Competition assays were conducted by flow cytometry according to themethods described. Hut-78 cells were harvested and stained in PBS 1×/BSA0.2%/EDTA 2 mM buffer for 1H at 4° C. using 5 μg/ml of increasingconcentrations of the antibodies, including 19H12, 12B11 and AZ158(previously identified) (0.006-200 μg/ml). After two washes, stainingdata were acquired on a BD FACSCanto II and analyzed using FlowJosoftware.

Increasing concentrations of (naked) 19H12, 12B11, and AZ158 were usedto shift labeled antibody bound to KIR3DL2 at the surface of HUT78 SScell lines. Antibody AZ158 (anti-D0 domain antibody) does not competewith the KIR3DL2 antibodies 19H12 or 12B11 for binding to KIR3DL2.

Antibodies were further tested for binding to a series of KIR3DL2mutants. Antibodies 19H12 and 12B11 did not show any loss of binding tounmutated wild-type KIR3DL2 (WTaKIR3DL2), but lost binding to mutant 11having P179T and S181T substitutions as well as to mutant 11A1 havingV178A and H1805 substitutions. The principal epitope of these antibodies19H12, 18B10 and 12B11 therefore includes residues P179, S181, V178and/or H180. These residues at positions 179 and 181 in mutant 11correspond to the residues present in KIR3DL1 (KIR3DL1 has T179 andT181). Residues P179 and S181 in particular are within the D1 domain ofKIR3DL2 and on the opposite face of the KIR3DL2 protein of theHLA-binding regions (i.e. the HLA binding pocket). Each of antibodies15C11, 19H12, 18B10 and 12B11 had reduced binding (full loss of bindingfor 15C11 and 19H12) to mutant M11A4 having substitutions E1305, H131Sand R145S. These residues at positions 179 and 181 in mutant 11correspond to the residues present in KIR3DL1 (KIR3DL1 has T179 andT181). Residues P179 and S181 in particular are within the D1 domain ofKIR3DL2 and on the opposite face of the KIR3DL2 protein of theHLA-binding regions (i.e., the HLA-binding pocket). Surface-exposedresidues adjacent to these mutated residues can also contribute to theepitopes of the antibodies, including for example residues N99, H100,E130, H131, F132, V178, H180, P182, Y183, and Q184 (reference to SEQ IDNO: 1) located at the surface of KIR3DL2 in the region of the P179/S181epitope but outside of the region of the KIR3DL2 mutations which did notresult in loss of binding of the antibodies (e.g., mutant 5 (residueP66) and mutant 8 (residue V127)).

Antibody 2B12 and other antibodies disclosed in the United States patentapplication publication 20150232556 had loss of binding to mutantshaving 160N and G62S substitutions and decrease in binding to mutantshaving P14S, S15A and H23S substitutions, but did not lose binding toany other mutants. The principal epitope of these antibodies thereforeincludes residues I60 and/or G62 (and the epitope optionally furtherincludes one or more of P14, S15, and H23). Residues 60 and 62 arewithin the D0 domain of KIR3DL2. Residues 14, 15, 23, 60 and 61 arewithin the D0 domain of KIR3DL2.

Example 2—Antibodies are Able to Kill KIR3DL2 Expressing Targets ViaAntibody-Dependent Cellular Cytotoxicity (ADCC)

Cell lysis through an ADCC mechanism was monitored in aradioactivity-based ⁵¹Cr release experiment (the level of radioactivityreleased from the preloaded target cells being proportional to theirdeath). One million target cells were loaded with ⁵¹Cr for 1 hour at 37°C. and washed 3 times. 3,000 cells were seeded per well (U-shaped bottom96-well plates) and test mAbs were added at 10 or 20 μg/ml finalconcentration (or increasing concentrations if dose-responserelationship is studied). Effector cells were added at a definedeffector:target ratio (in general 10:1) and the mixture was incubated at37° C. for 4 h. Supernatant is analyzed on a Lumaplate apparatus.

Anti-KIR3DL2 mAbs was tested at the same final concentration (10 μg/ml)to kill KIR3DL2-transfected B221 target cells. The mAbs, including19H12, were effective in mediating ADCC against KIR3DL2-expressing B221targets.

Example 3—Activity in Mouse Xenograft Models of KIR3DL2-Expressing HumanTumors

Tumor cell lines B221 and RAJI were made to express human KIR3DL2.Immune compromised mice used for B221-KIR3DL2 and RAJI-KIR3DL2 modelswere NOD-SCID purchased from Charles River Laboratories. In thefollowing models, 5 million human B221-KIR3DL2 or RAJI-KIR3DL2 tumorcells (in 100 μl PBS as vehicle) were engrafted IV on Day 0 (D0), i.e.,1 day before treatment initiation (D1). From D1, mice were treated IVwith different doses of anti-KIR3DL2 mAbs (doses were adapted to mousebody weight) diluted in PBS, 2 injections per week for the duration ofthe whole experiment.

Control groups included, depending on the experiment:

-   -   PBS/placebo-treated mice as a control of normal/unaffected tumor        growth; and    -   mice injected with the same dose of isotype control-matched mAbs        directed against an irrelevant antigen.

Mice were weighed and observed for clinical signs every 2 to 5 daysdepending on the model. Percent of body weight changes were calculatedas compared to body weight at D0 before tumor engraftment or to thehighest body weight reached during the experiment. Mouse deaths orimportant weight losses were recorded and used to draw survivalKaplan-Meier curves and calculate improvement in survival as compared tocontrol groups of mice.

The efficacy of IgG2b isotype murine anti-KIR3DL2 19H12 antibodies(given at 300 μg/mouse, twice a week) was separately tested against SCB221-KIR3DL2 xenografts or RAJI-KIR3DL2 xenografts (n=6 NOD-SCID miceper group). Animals treated with anti-KIR3DL2 antibodies showed anincrease in survival in comparison to mice treated with isotypecontrol-matched mAbs.

Example 4—Improved Detection Methods Reveal KIR3DL2-Positive Tumors

Tumor biopsies from RAJI-KIR3DL2 models and RAJI-KIR3DL2 cell lines wereobtained and staining was performed on frozen samples using AZ158antibody (see WO2010/081890) or antibodies 12B11 (see Example 1).KIR3DL2 was stained with anti-KIR3DL2 antibody by DAB chromogenicdetection according to standard protocols, adapted for immunostainingwith BenchMark XT (Ventana, Roche). For all staining control isotype(mIgG1) and control DAB were performed. Surprisingly, while AZ158 wasnegative, tumors were positive when using 12B11 antibody at the sameconcentration (5 μg/ml) of antibody (see FIG. 1). Raising concentrationsof antibody AZ158 (to 50 μg/ml) generated extensive background stainingthat did not allow tumor samples to be differentiated from healthytissue.

Next, tumor biopsies from cancer patients previously stained with AZ158were re-examined using antibody 12B11. Biopsies that had beenKIR3DL2-negative with AZ158 were stained with 12B11 (i.e., becomingKIR3DL2-positive). Results are shown in FIG. 2.

Example 5—KIR3DL2 is Expressed in PTCL

Tumor biopsies from PTCL patients were obtained and staining wasperformed on frozen samples. KIR3DL2 was stained with anti-KIR3DL2antibody 12B11 (mIgG1) by DAB chromogenic detection according tostandard protocols, adapted for immunostaining with BenchMark XT(Ventana, Roche). For all staining control isotype (mIgG1) and controlDAB were performed. CD30 was additionally stained. Tumors 3, 4 and 5were from the same patient. Tumors 1-5 are from patients having PTCL nototherwise specified. Tumors 6-8 are samples of mycosis fungoides, acutaneous T cell lymphoma (CTCL).

Results are shown in Table A below (LN=lymph node). Tumor samplecharacteristics are shown in Table B. PTCL from each of the samples fromthe patient from which tumor samples 3, 4 and 5 were obtained had strongmembranar staining, with a high percentage of cells beingKIR3DL2-positive. We also note that the patient from which samples 3-5were obtained had advanced (stage IV) disease while samples 1, 2, 6, 7and 8 represented less advanced disease (stage I or II); all had eitherno staining or low percentages of KIR3DL2+ tumor cells. It is possiblethat while some tumors are capable of expressing KIR3DL2 at high levelsand are thus suitable for targeting with a KIR3DL2 binding agent, tumorcells may acquire the NK marker KIR3DL2 at more advanced stages ofdisease, or more aggressive disease. KIR3DL2 may therefore be aparticularly suitable target for treatment of aggressive PTCLs and/oradvanced disease. Additionally, patients with earlier stages of diseasemay benefit from treatment or diagnostic assays using KIR3DL2 bindingagents to identify patients having prominent expression of KIR3DL2 onthe surface of tumor cells. Additionally, KIR3DL2-positive PTCL-NOStumors were found to include CD30 negative cases; KIR3DL2 may thereforefurthermore represent a therapeutic target when anti-CD30 antibodiescannot be used (or when tumors are resistant to anti-CD30 antibodies).

Example 6—KIR3DL2 is Expressed in Samples from ALCL and Ortho-VisceralExtranodal Disease (NK/T Lymphoma and EATL)

MEC04 and SNK6 NK/T lymphoma cells were stained for KIR3DL2 expressionusing flow cytometry (FACS), together with characterization of variouscell surface markers. KIR3DL2 was stained with anti-KIR3DL2 antibodylinked to phycoerythrin (PE). Additional markers evaluated were hCD56PE, hCD183/CXCR3 PE, hCD3 PE, hCD4 PE, hCD8 PE and CD54/ICAM PE. Cellswere harvested and stained using PE-labeled antibodies. After twowashes, stainings were acquired on a BD FACS Canto II and analyzed usingthe FlowJo software.

Results are shown in FIG. 3. Anti-KIR3DL2 antibody showed strongstaining on the MEC04 cells. MEC04 cells were additionally positive forstaining with CD183 (CXCR3), CD56 and CD54 (ICAM), but not CD3, CD4 orCD8 (the most common phenotype of extranodal NK/T lymphomas are surfaceCD3− and CD56+).

NK/T lymphoma cells, and in particular extranodal NK/T cell lymphoma,nasal type, can therefore express KIR3DL2 at significant levels, therebyproviding the possibility to treat such disease with anti-KIR3DL2antibodies. Additionally, KIR3DL2-positive NK/T lymphoma tumors werefound to express CD183 (CXCR3), CD56 and CD54 (ICAM), which may permitadministration of anti-KIR3DL2 in poor-prognosis patients, for examplethose having CXCR3 expression typically associated with poor diseaseprognosis.

Studies were carried out by immunohistochemistry (IHC) to provideconfirmation on patient samples and for different indications, bystaining primary tumor cells from human patients in frozen tissuesections with labeled anti-KIR3DL2 antibody. Briefly, cell lines knownto be positive and negative for KIR3DL2 expression were used as positiveand negative controls, respectively. In NK/T lymphomas, nasal type, 6patient samples were tested, of which 5 samples were interpretable. 2interpretable samples were positively stained and 3 were not, confirmingthat NK/T lymphomas express KIR3DL2. In samples from patients diagnosedwith enteropathy-associated T cell lymphoma (EATL), of 5 interpretablesamples, 2 were positively stained and 3 were negative for staining,confirming that EATL cells can express KIR3DL2. In samples from patientsdiagnosed with anaplastic large cell lymphoma (ALCL), of 4 interpretablepatient samples, 2 were positively stained and 2 were negative forstaining, confirming that ALCL cells can express KIR3DL2. Of the ALCLthat stained positive for KIR3DL2, samples included both ALK+ and ALK−.

TABLE A KIR3DL2 Tumor sample Staining CD30 staining Tumor 1: PositiveNegative LN/Lymphoma peripheral T cells Tumor 2: Negative NegativeTestis/Lymphoma peripheral T cells Tumor 3: Negative PositiveSpleen/Lymphoma peripheral T cells Tumor 4: Negative PositiveSpleen/Lymphoma peripheral T cells Tumor 5: Negative PositiveSpleen/Lymphoma peripheral T cells Tumor 6: Positive Positive LN/Mycosisfungoides Tumor 7: Positive Positive LN/Mycosis fungoides Tumor 8:Positive Positive LN/Mycosis fungoides

TABLE B Tumor stage % % % Sample Tissue type Appearance Pathologydiagnosed (minimum) normal % lesion tumor nercosis 1 Lymphoidtissue/lymphatic Tumoral PTCL (unspecified) I 5 0 90 0 ganglion 2Lymphoid tissue/testicle Tumoral PTCL-NOS (unspecified) IE 10 0 60 0 3Lymphoid tissue/spleen Tumoral PTCL-NOS (unspecified) IV 10 0 40 0 4Lymphoid tissue/spleen Tumoral PTCL-NOS (unspecified) IV 5 0 90 0 5Lymphoid tissue/spleen Tumoral PTCL-NOS (unspecified) IV 50 0 50 0 6Lymphoid tissue/lymphatic Tumoral Mycosis fungoides II 0 0 70 20ganglion 7 Lymphoid tissue/lymphatic Tumoral Mycosis fungoides II 0 0 8010 ganglion 8 Lymphoid tissue/lymphatic Tumoral Mycosis fungoides II 0 080 10 ganglion

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a”, “an”, “the” and similar referents in thecontext of describing the invention is to be construed to cover both thesingular and the plural, unless otherwise indicated herein or clearlycontradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about”, where appropriate).

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including”, or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The use of any and all examples or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

We claim:
 1. A method of treating an enteropathy-associated T celllymphoma (EATL), comprising administering to an individual sufferingfrom said EATL a therapeutically active amount of an antibody that bindsa KIR3DL2 polypeptide and that further: i) directs ADCC toward aKIR3DL2-expressing cell, and/or ii) delivers a cytotoxic agent to aKIR3DL2-expressing cell.
 2. The method of claim 1, wherein the treatmentcomprises: a) determining the KIR3DL2 polypeptide status of malignantcells within the individual having an EATL, and b) upon a determinationthat the individual has KIR3DL2 polypeptide expressed on the surface ofmalignant cells, administering to the individual said antibody thatbinds a KIR3DL2 polypeptide and that further: i) directs ADCC toward theKIR3DL2-expressing malignant cells and/or ii) delivers a cytotoxic agentto the KIR3DL2-expressing malignant cells.
 3. The method of claim 1,wherein the anti-KIR3DL2 antibody directs ADCC toward aKIR3DL2-expressing cell.
 4. The method of claim 3, wherein the antibodycomprises an amino acid modification that enhances binding to a humanFcγ receptor.
 5. The method of claim 1, wherein the antibody is linkedto a cytotoxic agent.
 6. The method of claim 3, wherein the anti-KIR3DL2antibody binds human KIR3DL2 but does not bind to human KIR3DL1.
 7. Themethod of claim 3, wherein the anti-KIR3DL2 antibody has reduced bindingto a KIR3DL2 polypeptide having a mutation at residue P179 and/orresidue S181, compared to a wild-type KIR3DL2 polypeptide of SEQ IDNO:
 1. 8. The method of claim 3, wherein the anti-KIR3DL2 antibody hasreduced binding to a KIR3DL2 polypeptide having a mutation at residueI60 and/or residue G62, compared to a wild-type KIR3DL2 polypeptide ofSEQ ID NO:
 1. 9. The method of claim 1, wherein the anti-KIR3DL2antibody binds human KIR3DL2 but does not bind to human KIR3DL1.
 10. Themethod of claim 1, wherein the anti-KIR3DL2 antibody has reduced bindingto a KIR3DL2 polypeptide having a mutation at residue P179 and/orresidue S181, compared to a wild-type KIR3DL2 polypeptide of SEQ IDNO:
 1. 11. The method of claim 1, wherein the anti-KIR3DL2 antibody hasreduced binding to a KIR3DL2 polypeptide having a mutation at residueI60 and/or residue G62, compared to a wild-type KIR3DL2 polypeptide ofSEQ ID NO: 1.